Nucleotide and deduced amino acid sequences of tumor gene Int6

ABSTRACT

The present invention discloses the isolation of the human and murine wild-type Int6 gene and the cDNAs corresponding to these genes. The invention further describes the use of reagents derived from the nucleic acid and amino acid sequences of the Int6 gene in diagnostic methods, immunotherapy, gene therapy and as vaccines.

[0001] This is a continuation-in-part application of U.S. Ser. No.08/385,998, filed Feb. 9, 1995.

FIELD OF INVENTION

[0002] The present invention relates to the area of cancer diagnosticsand therapeutics. More specifically, the invention relates to the Int6gene and to the use of reagents derived from the nucleic acid anddeduced amino acid sequences of the Int6 gene in gene therapy, vaccines,diagnostic methods and immunotherapy.

BACKGROUND OF INVENTION

[0003] The mouse mammary tumor virus (MMTV) is a retrovirus which hasbeen shown to act as an insertional mutagen that causes the deregulationof expression of cellular genes adjacent to the site of MMTV integrationin mammary tumors (Varmus, H. E. (1982). Cancer Surv., 1:309-320). Miceinfected with MMTV frequently develop preneoplastic hyperplasticalveolar nodules (HAN) (Daniel, C., et al. Proc. Natl. Acad. Sci. USA.,61:53-60; DeOme, K. B., (1959) J. Natl. Cancer Inst., 78:751-757;Medina, D., (1973) Methods Cancer Res., 7:3-53; Smith, G., et al. (1984)Cancer Res., 44:3426-3437) and the passage of these nodules in clearedmammary fat pads of syngeneic mice by serial outgrowth often results inthe development of mammary tumors within these mice in a stochasticmanner. In addition, it is not uncommon to find metastatic lesions inthe lungs of mice bearing outgrowths with mammary tumors. For thesereasons, there is considerable interest in identifying MMTV-inducedmutational events that may contribute to different stages of tumordevelopment.

[0004] Using the MMTV genome as a molecular tag, five loci (Wnt-1/Int-1,Fgf-3/Int-2, Int-3, Wnt-3, and Fgf-4/Hst/k-FGF) have been identifiedwhich represent common integration sites (designated Int loci) for MMTVin mouse mammary tumors (Dickson, C., et al. (1984) Cell., 37:529-536;Gallahan, D., et al. (1987) J. Virol., 61:218-220; Nusse, R., et al.(1982) Cell., 31:99-109; Peters, G., et al. (1989) Proc. Natl. Acad.Sci. USA., 86:5678-5682). Transgenic mouse studies utilizing transgenesin which the MMTV LTR was linked to either the WNT-1, Fgf-3, or Int-3genes have demonstrated that activation of expression of these genescontributes to mammary tumorigenesis (Jhappan, C., et al. (1992) Genes &Develop., 6:345-355; Muller, W. J., et al. (1990) Embo J., 9:907-913;Tsukamoto, A. S., et al. (1988) Cell., 55:619-625).

[0005] The present invention describes the isolation of a new Int genedesignated Int6 and the use of the gene, its gene product, and reagentsderived from the gene and its gene product, in diagnostic methods,vaccines, immunotherapy and gene therapy.

SUMMARY OF INVENTION

[0006] The present invention relates to the isolation of the Int6 gene.The invention also relates to the murine and human cDNAs which comprisethe coding sequence of the Int6 gene. The invention further relates tonucleic acid sequences derived from the Int6 gene and the Int6 cDNAs andthe use of these nucleic acid sequences as probes to isolate homologuesof the Int6 gene in other mammals or as probes to detect mutations ofthe Int6 gene.

[0007] It is also an object of the present invention to providesynthetic nucleic acid sequences capable of directing production ofrecombinant Int6 protein and peptide fragments derived therefrom, aswell as equivalent natural nucleic acid sequences. Such natural nucleicacid sequences may be isolated from a cDNA or genomic library from whichthe gene capable of directing the synthesis of the Int6 protein may beidentified and isolated. For the purposes of this application, nucleicacid sequence refer to RNA, DNA, cDNA or any synthetic variant thereof.

[0008] The present invention further relates to Int6 protein andpeptides derived therefrom.

[0009] The invention also relates to antibodies directed against Int6protein or peptides derived therefrom.

[0010] The invention also provides methods for detecting mutations ofInt6 gene where detection of such mutations is useful in determining thepresence of a neoplastic tissue in a subject or a genetic predispositionto cancer in a subject.

[0011] A first method for detecting mutations of the Int6 gene comprisesanalyzing DNA of a subject for mutations of the Int6 gene.

[0012] A second method for detecting mutations of the Int6 genecomprises analyzing RNA of a subject for alterations in the Int6 mRNAexpression.

[0013] Yet another method for detecting mutations of the Int6 genecomprises analyzing protein of a subject for alterations in Int6 proteinexpression.

[0014] The present invention also provides pharmaceutical compositionsfor use as vaccines for immunizing a subject against cancer and for usein immunotherapy methods. One such composition comprises nucleic acidsequence capable of directing host organism synthesis of Int6 protein orpeptide fragments thereof while a second pharmaceutical compositioncomprises Int6 protein or peptides derived therefrom.

[0015] The above pharmaceutical compositions may also be used inimmunotherapy methods for treating a subject having cancer. For use inimmunotherapy, the present invention further provides a thirdpharmaceutical composition comprising antibodies directed against Int6protein or peptides derived therefrom where such antibodies are coupledto toxin molecules, radioisotopes or drugs.

[0016] The present invention therefore relates to application ofimmunotherapy to subjects having cancer comprising administering one ormore of the above pharmaceutical compositions to said subject in atherapeutically effective amount.

[0017] The invention further relates to a method for treating a subjecthaving cancer comprising:

[0018] (a) immunizing the subject with an amount of an expression vectorencoding Int6 protein or with Int6 protein itself, said amount effectiveto elicit a specific T cell response;

[0019] (b) isolating said T cells from said immunized subject; and

[0020] (c) administering said T cells to said immunized subject or to anunimmunized subject in a therapeutically effective amount.

[0021] The invention also provides a diagnostic kit for determining thenucleotide sequence of Int6 alleles by the polymerase chain reaction,said kit comprising purified and isolated nucleic acid sequences usefulas PCR primers. These PCR primers are also useful in analyzing DNA orRNA of a subject for mutations of the Int6 gene.

[0022] The present invention further provides a method for supplying thewild-type Int6 gene to a cell having altered expression of the Int6protein by virtue of a mutation in the Int6 gene, the method comprising:introducing a wild-type Int6 gene into a cell having altered expressionof the Int6 protein such that said wild-type gene is expressed in thecell.

DESCRIPTION OF FIGURES

[0023]FIGS. 1A and 1B show the results of Southern blot analyses inwhich 10 micrograms of cellular DNA was digested by EcoRI, separated byagarose gel electrophoresis, and hybridized with MMTV LTR probe. In FIG.1A, the DNAs analyzed were isolated from a CZZ-1 pre-neoplastichyperplastic outgrowth line designated CZZ-1 HOG (lane 1) and from CZZ-1derived mammary tumors 22 (lane 2), 1262 (lane 3), 1263 (lane 4), 20(lane 5), 19 (lane 6), 23 (lane 7), 21 (lane 8), 24 (lane 9), 8 (lane10), 9 (lane 11), 12 (lane 12), 13 (lane 13) and 14 (lane 14) asindicated at the top of FIG. 1A. In FIG. 1B, the DNAs analyzed wereisolated from CZZ-1 derived tumor 4973 (lane 1) and from 11 independentlung metastasis (lanes 2-12) from a mouse bearing tumor 4973 asindicated at the top of FIG. 1B.

[0024] FIGS. 2A-2C show the results of Southern blot analyses of 10micrograms of cellular DNA digested with EcoRI, separated by agarose gelelectrophoresis and hybridized as follows. Lanes 1 and 2 of the blots inFIGS. 2A and 2B were hybridized with probe D which corresponds to hostflanking sequences. Lane 2 of the blots in FIGS. 2A and 2B wassubsequently hybridized with MMTV gag sequences (a 2.0 kb-Pst-XhoIfragment from MMTV GR-40; Gallahan and Callahan J. Virol, 61, 66-74:(1987)) and the results are shown in lane 3 in FIGS. 2A and 2B. Lanes 1and 2 in FIG. 2C were hybridized with probe C. Lane 2 was subsequentlyhybridized with MMTV env sequences (1.7 kb PstI fragment from MMTV(C3H); Gallahan and Callahan J. Virol, 61: 66-74; (1987)) and the resultis shown in lane 3 in FIG. 2C. The DNAs analyzed were isolated fromCZZ-1 HOG derived tumor 22 (FIG. 2A, lanes 2 and 3), independent mammarytumor 1139 (FIG. 2B, lanes 2 and 3) independent mammary tumor 3134 (FIG.2C, lanes 2 and 3), and normal liver (Lane 1 in FIGS. 2A-2C). Thepresence of the arrow in FIGS. 2A-2C indicates the site of the MMTVinduced rearranged restriction fragment. The locations of probes C and Din the Int6 gene are shown in FIG. 3.

[0025]FIG. 3 is a schematic diagram of the murine Int6 locus in whichthe location of the four overlapping lambda clones which span the Intolocus (designated 1 to 4) are shown relative to a partial restrictionmap of sites for EcoRI (E), XbaI (X), PstI (P), BglII (BGL), HindIII(H), and BamHI (B) in the Int6 locus. The location of the Int6 exons andof restriction fragments used as probes A-D are indicated below therestriction map by solid and hatched boxes respectively. The scale ofthe restriction map is given in increments of 1.0 kb at the bottom ofthe Figure while the location and transcriptional orientation ofintegrated MMTV proviral genomes within the Int6 gene of tumors 1139 and3144, or within the Int6 gene of the CZZ-1 HOG, is indicated byarrowheads above the restriction map.

[0026]FIG. 4 shows the results of Northern blot analysis of total RNAisolated from Int6 negative tumor (tumor 178) and the Int6 positivetumors (22 and 1139). The RNAs were denatured in the presence offormaldehyde, separated on a 1% agarose gel containing formaldehyde, andhybridized with probe A.

[0027]FIG. 5 shows the complete nucleotide sequence of the murine 1.4 kbInt6 cDNA where intron breaks are indicated by small arrows (▴) abovethe start of the next exon and the deduced amino acid sequence of thegene product is given below the nucleotide sequence. Potentialphosphorylation sites for cyclic AMP/cyclic GMP-dependent protein kinase(∘), protein kinase C (Δ), tyrosine kinases (−), casein kinase II (∇),and glycosylation sites (□) are indicated in the deduced amino acidsequence. Abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp;E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; M. Met; N, Asn; P, Pro;Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

[0028]FIG. 6 shows the results of a “Zoo” blot in which cellular DNA (10μg each) isolated from C. Elegans, Drosophila, Xenopus, chicken, mouseand human was hybridized with Int6 cDNA under high stringencyconditions.

[0029]FIG. 7 shows the results of Northern blot analyses of total RNA(10 μg each) isolated from normal adult tissues (top panel) and fromdeveloping embryos (lower panel). The RNAs were denatured in thepresence of formaldehyde, run on 1% agarose gels containing formaldehydeand then transferred to a nylon membrane and hybridized sequentiallywith a β-actin probe (the 2.3 kb mRNAs) and then a murine Int6 cDNAprobe (the 1.4 kb mRNAs).

[0030]FIG. 8 shows a schematic illustrating the location and nucleotidesequence of a cryptic transcription stop signal (underlined) in thereverse sequence of the MMTV LTR where the sequence shown corresponds tobases 464 through 497 from the three prime end of the LTR. The hatchedbox corresponds to an Int6 exon and the open box corresponds to the LTRof an integrated MMTV proviral genome integrated within Int6. The U5, Rand U3 regions of the MMTV LTR are indicated as well as thetranscriptional orientations of the MMTV genome and the Int6 gene.

[0031]FIGS. 9A and 9B show the nucleotide sequences of the junctionsbetween MMTV and Int6 sequences in chimeric Int6-MMTV LTR RNA speciesdetected in tumors 1139 (FIG. 9A) and 22 (FIG. 9B). In FIG. 9A, thenucleotide sequence shown begins at the 5′ end of exon with thenucleotide sequence shown in lower case letters corresponding to intron5 sequences. Amino acid sequences in RNA species 2 which are identicalto those in RNA species 1 are indicated by dots and nucleotide sequenceswhich have been spliced out in RNA species 2 are indicated by dashes.Nucleotide and amino acid sequences which are underlined are from theintegrated MMTV genome.

[0032] In FIG. 9B, the nucleotide sequence shown for the chimeric RNAspecies detected in tumor 22 begins at the 5′ end of exon 9 and runthrough a portion of intron 9 to the cryptic poly A addition signal inthe MMTV genome. Intron nucleotide sequence is given in lower caseletters, MMTV sequences are underlined, and dashes indicate nucleotidesequences of the intron which have been spliced out. Dots correspond toamino acid residues encoded by RNA species 2 and 3 which are identicalto those encoded by RNA species 1. The abbreviations for amino acidsshown in FIGS. 9A and 9B are the same as those given in the legend forFIG. 5.

[0033]FIG. 10 shows the genomic map of the human Int6 gene. As in themouse genome, the human Int6 gene is composed of 13 exons (filled bars)where the nucleotide boundaries for each exon are presented in theFigure. The location of a CA-repeat sequence in the seventh intron isalso shown.

[0034]FIG. 11 shows the nucleotide sequence of primers (underlined)complementary to the nucleic acid sequences (not underlined) flankingthe CA-repeat in intron 7 of the human Int6 gene. The distance of theprimers from the CA-repeat are presented as 40 and 138 base pairsrespectively and the number of CA-repeats (18) shown is that found inthe wild-type Int6 gene. The upper nucleic acid sequences are shown inthe 5′ to 3′ orientation and the lower nucleic acid sequences are shownin the 3′ to 5′ orientation reading left to right.

[0035]FIG. 12 shows the results of Southern blots in which human DNA(lane 4), Chinese hamster-human somatic cell hybrid DNA containing onlyhuman chromosome 6 (lane 1) or human chromosome 8 (lane 2) andmouse-human somatic cell hybrid DNA which contains human chromosomes3,7,8,15 and 17 (lane 3) were digested with Hind III and hybridized withhuman Int6 cDNA.

[0036]FIG. 13 shows the results of PCR amplification of DNA from primaryhuman breast tumor (T) and matching normal tissue (L, where L indicateslymphocyte) using labelled primers flanking the CA repeat sequence inintron 7 of the Int6 gene. The numbers above each blot representpatients from which tumor and matching normal tissue samples wereobtained. The arrow next to each blot indicates the complete loss, or asignificantly reduced signal, of one allele relative to the matchingnormal DNA.

[0037]FIG. 14 shows the nucleotide sequences bounding 12 of the 13 humanInt6 exons. Nucleotide sequences of primers (underlined) complementaryto nucleic acid sequences (not underlined) bounding each exon are shownto the left and right of each exon. The upper nucleic acid sequencesshown to the left and right of each exon are in the 5′ to 3′ orientationwhile lower sequences shown to the left and right of each exon are inthe 3′ to 5′ orientation. When the sequences bounding each exon do notbegin at the intron-exon junction, the distance of the sequence from thejunction is given in base pairs to the left or right of each exon.

[0038]FIG. 15 shows the results of a Northern blot of total RNA (20 μgeach) isolated from primary breast tumor (T) and matching normal (N)tissue. The RNA samples were denatured in the presence of formaldehyde,run on 1% agarose gels containing formaldehyde, then transferred to anylon membrane and hybridized sequentially with a actin-probe and ahuman Int6 cDNA probe.

[0039]FIG. 16 shows the results of a Northern blot of total RNA (20 μgeach) isolated from human nonsmall cell lung carcinomas (54T and 55T)and matching normal tissue (55N). Matching normal tissue for 54T wasalso surveyed for Int6 mRNA expression but the Northern blot for 54N isnot shown. Northern blot analysis was carried out as described in FIG.15.

DESCRIPTION OF INVENTION

[0040] The present invention discloses that mutational events associatedwith MMTV integration into the host cell genome in tumorigenesis occurin a previously unknown gene designated Int6. This gene is located onmouse chromosome 15. More specifically, the present invention relates toInt6 gene and its corresponding cDNA. The region of mouse chromosome 15containing the Int6 is represented in the four overlapping lambda clones(designated 1-4 in FIG. 3) and comprises the entire Int6 gene. The Int6gene represents the wild-type Int6 gene.

[0041] The present invention is also directed to the full-length cDNAcorresponding to the murine Int6 gene. This cDNA sequence is set forthbelow as SEQ ID NO:1. AACAAGCGCT CCTTTCCCCC GGCAAGATGG CGGAGTACGA 40CTTGACTACT GCATCGCGCA TTTTCTGGAT CGGCACCTGG 80 TCTTTCCGCT TCTTGAGTTTCTCTCTGTGA AAGAGATTTA 120 TAATGAAAAA GAATTATTAC AAGGAAAATT AGATCTTCTT160 AGTGATACCA ATATGGTGGA CTTTGCTATG GATGTTTACA 200 AAAACCTTTATTCTGATGAT ATCCCTCATG CTTTGAGAGA 240 AAAAAGAACC ACAGTTGTTG CGCAGCTGAAACAGCTCCAG 280 GCAGAAACAG AACCAATTGT GAAGATGTTT GAAGATCCAG 320AAACTACAAG GCAGATGCAG TCAACCAGGG ATGGCAGGAT 360 GTTATTTGAC TACCTGGCAGACAAACATGG GTTTAGGCAA 400 GAGTACTTAG ATACACTCTA CAGATACGCA AAATTCCAGT440 ATGAGTGTGG AAATTACTCT GGAGCTGCAG AGTATCTTTA 480 CTTCTTTAGAGTTTTGGTCC CAGCAACAGA TAGAAATGCT 520 TTAAGTTCGC TCTGGGGAAA ACTGGCCTCTGAAATCTTAA 560 TGCAGAATTG GGATGCAGCC ATGGAAGACC TTACTCGATT 600AAAAGAAACC ATAGACAATA ATTCTGTGAG TTCTCCACTC 640 CAGTCTCTTC AGCAGCGAACATGGCTCATT CATTGGTCTC 680 TATTTGTTTT TTTCAACCAT CCAAAGGGCC GTGATAACAT720 TATTGATCTC TTCCTTTACC AACCACAGTA TCTTAATGCA 760 ATTCAGACAATGTGTCCACA TATTCTACGC TATTTGACTA 800 CTGCCGTCAT AACCAACAAA GATGTGCGGAAACGCCGGCA 840 GGTGCTGAAA GATCTGGTGA AAGTGATTCA ACAGGAGTCT 880TACACATATA AAGACCCAAT TACAGAATTT GTTGAATGCC 920 TATATGTTAA CTTTGATTTTGACGGGGCTC AGAAAAAGCT 960 GAGAGAATGT GAATCAGTGC TCGTGAATGA CTTCTTCCTG1000 GTAGCGTGTC TGGAGGACTT CATTGAGAAT GCCCGTCTCT 1040 TCATATTTGAGACGTTTTGT CGTATCCACC AGTGTATCAG 1080 CATTAATATG TTAGCAGATA AACTGAATATGACTCCAGAA 1120 GAAGCTGAAA GATGGATTGT GAATTTGATT AGAAATGCGA 1160GGTTGGATGC CAAGATTGAT TCTAAACTAG GTCATGTGGT 1200 AATGGGCAAC AATGCAGTCTCGCCCTACCA GCAAGTGATT 1240 GAAAAGACCA AAAGCCTTTC TTTTAGAAGC CAAATGTTGG1280 CCATGAATAT TGAAAAGAAA CTTAATCAGA ACAGTAGATC 1320 AGAGGCTCCCAACTGGGCAA CCCAAGACTC TGGCTTCTAT 1360 TAAAGGATTA TAAAGAAAAG AAGAAAAAGGAATAAGTGAA 1400 AGACACAGTA GCCATTGTGT ATAAAGGATG ACATACATTT 1440TTAGAAGCAA TTAACATGTT TGCTACAAAT TTTGGAGAAT 1480 TTGAATAAAA TTGGCTATGATTAA 1504

[0042] The abbreviation used for the nucleotides are those standardlyused in the art.

[0043] The deduced amino acid sequence of the murine Int6 cDNA is shownas SEQ ID NO:2 below and starts at nucleotide 173 of SEQ ID NO:1 andextends 1188 nucleotides. Met Val Asp Phe Ala Met Asp Val Tyr Lys AsnLeu   1               5                  10 Tyr Ser Asp Asp Ile Pro HisAla Leu Arg Glu Lys          15                  20 Arg Thr Thr Val ValAla Gln Leu Lys Gln Leu Gln  25                  30                  35Ala Glu Thr Glu Pro Ile Val Lys Met Phe Glu Asp             40                  45 Pro Glu Thr Thr Arg Gln Met Gln SerThr Arg Asp      50                  55                  60 Gly Arg MetLeu Phe Asp Tyr Leu Ala Asp Lys His                 65                  70 Gly Phe Arg Gln Glu Tyr Leu AspThr Leu Tyr Arg          75                  80 Tyr Ala Lys Phe Gln TyrGlu Cys Gly Asn Tyr Ser  85                  90                  95 GlyAla Ala Glu Tyr Leu Tyr Phe Phe Arg Val Leu            100                 105 Val Pro Ala Thr Asp Arg Asn Ala LeuSer Ser Leu     110                 115                 120 Trp Gly LysLeu Ala Ser Glu Ile Leu Met Gln Asn                125                 130 Trp Asp Ala Ala Met Giu Asp LeuThr Arg Leu Lys         135                 140 Glu Thr Ile Asp Asn AsnSer Val Ser Ser Pro Leu 145                 150                 155 GlnSer Leu Gln Gln Arg Thr Trp Leu Ile His Trp            160                 165 Ser Leu Phe Val Phe Phe Asn His ProLys Gly Arg     170                 175                 180 Asp Asn IleIle Asp Leu Phe Leu Tyr Gln Pro Gln                185                 190 Tyr Leu Asn Ala Ile Gln Thr MetCys Pro His Ile         195                 200 Leu Arg Tyr Leu Thr ThrAla Val Ile Thr Asn Lys 205                 210                 215 AspVal Arg Lys Arg Arg Gln Val Leu Lys Asp Leu            220                 225 Val Lys Val Ile Gln Gln Glu Ser TyrThr Tyr Lys     230                 235                 240 Asp Pro IleThr Glu Phe Val Glu Cys Leu Tyr Val                245                 250 Asn Phe Asp Phe Asp Gly Ala GlnLys Lys Leu Arg         255                 260 Glu Cys Glu Ser Val LeuVal Asn Asp Phe Phe Leu 265                 270                 275 ValAla Cys Leu Glu Asp Phe Ile Glu Asn Ala Arg            280                 285 Leu Phe Ile Phe Glu Thr Phe Cys ArgIle His Gln     290                 295                 300 Cys Ile SerIle Asn Met Leu Ala Asp Lys Leu Asn                305                 310 Met Thr Pro Glu Glu Ala Glu ArgTrp Ile Val Asn         315                 320 Leu Ile Arg Asn Ala ArgLeu Asp Ala Lys Ile Asp 325                 330                 335 SerLys Leu Gly His Val Val Met Gly Asn Asn Ala            340                 345 Val Ser Pro Tyr Gln Gln Val Ile GluLys Thr Lys     350                 355                 360 Ser Leu SerPhe Arg Ser Gln Met Leu Ala Met Asn                365                 370 Ile Glu Lys Lys Leu Asn Gln AsnSer Arg Ser Glu         375                 380 Ala Pro Asn Trp Ala ThrGln Asp Ser Gly Phe Tyr 385                 390                 395

[0044] The present invention also discloses the nucleotide sequence ofthe human homologue of the Int6 cDNA where the human Int6 cDNA sequenceis 89% homologous to the mouse sequence. Two overlapping recombinantclones (HINT6A and HINT6B) represent the 5′ and 3′ halves respectivelyof the human Int6 cDNA sequence. These two clones were deposited withthe American Type Culture collection (ATCC), 12301 Parklawn Drive,Parkville, Md. 20852 on Jan. 24, 1995 and have ATCC accession numbers97029 and 97030. The human Int6 cDNA sequence is shown below as SEQ IDNO:3. ACTCCCTTTT CTTTGGCAAG ATGGCGGAGT ACGACTTGAC 40 TACTCGCATCGCGCACTTTT TGGATCGGCA TCTAGTCTTT 80 CCGCTTCTTG AATTTCTCTC TGTAAAGGAGATATATAATG 120 AAAAGGAATT ATTACAAGGT AAATTGGACC TTCTTAGTGA 160TACCAACATG GTAGACTTTG CTATGGATGT ATACAAAAAC 200 CTTTATTCTG ATGATATTCCTCATGCTTTG AGAGAGAAAA 240 GAACCACAGT GGTTGCACAA CTGAAACAGC TTCAGGCAGA280 AACAGAACCA ATTGTGAAGA TGTTTGAAGA TCCAGAAACT 320 ACAAGGCAAATGCAGTCAAC CAGGGATGGT AGGATGCTCT 360 TTGACTACCT GGCGGACAAG CATGGTTTTAGGCAGGAATA 400 TTTAGATACA CTCTACAGAT ATGCAAAATT CCAGTACGAA 440TGTGGGAATT ACTCAGGAGC AGCAGAATAT CTTTATTTTT 480 TTAGAGTGCT GGTTCCAGCAACAGATAGAA ATGCTTTAAG 520 TTCACTCTGG GGAAAGCTGG CCTCTGAAAT CTTAATGCAG560 AATTGGGATG CAGCCATGGA AGACCTTACA CGGTTAAAAG 600 AGACCATAGATAATAATTCT GTGAGTTCTC CACTTCAGTC 640 TCTTCAGCAG AGAACATGGC TCATTCACTGGTCTCTGTTT 680 GTTTTCTTCA ATCACCCCAA AGGTCGCGAT AATATTATTG 720ACCTCTTCCT TTATCAGCCA CAATATCTTA ATGCAATTCA 760 GACAATGTGT CCACACATTCTTCGCTATTT GACTACAGCA 800 GTCATAACAA ACAAGGATGT TCGAAAACGT CGGCAGGTTC840 TAAAAGATCT AGTTAAAGTT ATTCAACAGG AGTCTTACAC 880 ATATAAAGACCCAATTACAG AATTTGTTGA ATGTTTATAT 920 GTTAACTTTG ACTTTGATGG GGCTCAGAAAAAGCTGAGGG 960 AATGTGAATC AGTGCTTGTG AATGACTTCT TCTTGGTGGC 1000TTGTCTTGAG GATTTCATTG AAAATGCCCG TCTCTTCATA 1040 TTTGAGACTT TCTGTCGCATCCACCAGTGT ATCAGCATTA 1080 ACATGTTGGC AGATAAATTG AACATGACTC CAGAAGAAGC1120 TGAAAGGTGG ATTGTAAATT TGATTAGAAA TGCAAGACTG 1160 GATGCCAAGATTGATTCTAA ATTAGGTCAT GTGGTTATGG 1200 GTAACAATGC AGTCTCACCC TATCAGCAAGTGATTGAAAA 1240 GACCAAAAGC CTTTCCTTTA GAAGCCAGAT GTTGGCCATG 1280AATATTGAGA AGAAACTTAA TCAGAATAGC AGGTCAGAGG 1320 CTCCTAACTG GGCAACTCAAGATTCTGGCT TCTACTGAAG 1360 AACCATAAAG AAAAGATGAA AAAAAAAACT ATCAAAGAAA1400 GATGAAATAA TAAAACTATT ATATAAAGGG TGACTTACAT 1440 TTTGGAAACAACATATTACG TATAAATTTT GAAGAATTGG 1480 AATAAAATTG ATTCATTTTA 1500

[0045] The deduced amino acid sequence of the human Int6 cDNA is shownbelow as SEQ ID NO:4 and starts at nucleotide 168 of SEQ ID NO:3 andextends 1188 nucleotides. Met Val Asp Phe Ala Met Asp Val Tyr Lys AsnLeu   1               5                  10 Tyr Ser Asp Asp Ile Pro HisAla Leu Arg Glu Lys          15                  20 Arg Thr Thr Val ValAla Gln Leu Lys Gln Leu Gln  25                  30                  35Ala Glu Thr Glu Pro Ile Val Lys Met Phe Glu Asp             40                  45 Pro Glu Thr Thr Arg Gln Met Gln SerThr Arg Asp      50                  55                  60 Gly Arg MetLeu Phe Asp Tyr Leu Ala Asp Lys His                 65                  70 Gly Phe Arg Gln Glu Tyr Leu AspThr Leu Tyr Arg          75                  80 Tyr Ala Lys Phe Gln TyrGlu Cys Gly Asn Tyr Ser  85                  90                  95 GlyAla Ala Glu Tyr Leu Tyr Phe Phe Arg Val Leu            100                 105 Val Pro Ala Thr Asp Arg Asn Ala LeuSer Ser Leu     110                 115                 120 Trp Gly LysLeu Ala Ser Glu Ile Leu Met Gln Asn                125                 130 Trp Asp Ala Ala Met Glu Asp LeuThr Arg Leu Lys         135                 140 Glu Thr Ile Asp Asn AsnSer Val Ser Ser Pro Leu 145                 150                 155 GlnSer Leu Gln Gln Arg Thr Trp Leu Ile His Trp            160                 165 Ser Leu Phe Val Phe Phe Asn His ProLys Gly Arg     170                 175                 180 Asp Asn IleIle Asp Leu Phe Leu Tyr Gln Pro Gln                185                 190 Tyr Leu Asn Ala Ile Gln Thr MetCys Pro His Ile         195                 200 Leu Arg Tyr Leu Thr ThrAla Val Ile Thr Asn Lys 205                 210                 215 AspVal Arg Lys Arg Arg Gln Val Leu Lys Asp Leu            220                 225 Val Lys Val Ile Gln Gln Glu Ser TyrThr Tyr Lys     230                 235                 240 Asp Pro IleThr Glu Phe Val Glu Cys Leu Tyr Val                245                 250 Asn Phe Asp Phe Asp Gly Ala GlnLys Lys Leu Arg         255                 260 Glu Cys Glu Ser Val LeuVal Asn Asp Phe Phe Leu 265                 270                 275 ValAla Cys Leu Glu Asp Phe Ile Glu Asn Ala Arg            280                 285 Leu Phe Ile Phe Glu Thr Phe Cys ArgIle His Gln     290                 295                 300 Cys Ile SerIle Asn Met Leu Ala Asp Lys Leu Asn                305                 310 Met Thr Pro Glu Glu Ala Glu ArgTrp Ile Val Asn         315                 320 Leu Ile Arg Asn Ala ArgLeu Asp Ala Lys Ile Asp 325                 330                 335 SerLys Leu Gly His Val Val Met Gly Asn Asn Ala            340                 345 Val Ser Pro Tyr Gln Gln Val Ile GluLys Thr Lys     350                 355                 360 Ser Leu SerPhe Arg Ser Gln Met Leu Ala Met Asn                365                 370 Ile Glu Lys Lys Leu Asn Gln AsnSer Arg Ser Glu         375                 380 Ala Pro Asn Trp Ala ThrGln Asp Ser Gly Phe Tyr 385                 390                 395

[0046] The amino acid sequence of human and mouse Int6 proteins areidentical.

[0047] Variations are contemplated in the cDNA sequences shown in SEQ IDNOs:1 and 3 which will result in a DNA sequence that is capable ofdirecting production of analogs of the proteins shown in SEQ ID NOs:2and 4 respectively. It should be noted that the DNA sequences set forthabove represents a preferred embodiment of the present invention. Due tothe degeneracy of the genetic code, it is to be understood that numerouschoices of nucleotides may be made that will lead to a DNA sequencecapable of directing production of the instant Int6 proteins or theiranalogs. As such, DNA sequences which are functionally equivalent to thesequence set forth above or which are functionally equivalent tosequences that would direct production of analogs of the Int6 proteinsproduced pursuant to the amino acid sequences set forth above, areintended to be encompassed within the present invention.

[0048] The term analog includes any protein or polypeptide having anamino acid residue sequence substantially identical to a sequencespecifically shown herein in which one or more amino acid residues havebeen conservatively substituted with a functionally similar residue.Examples of conservative substitutions include, for example, thesubstitution of one non-polar (i.e. hydrophobic) residue such asisoleucine, valine, leucine or methionine for another; the substitutionof one polar (i.e. hydrophilic) residue for another, such as asubstitution between arginine and lysine, between glutamine andasparagine, or between glycine and serine; the substitution of one basicresidue such as lysine, arginine or histidine for another; or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another.

[0049] The phrase conservative substitution may also include the use ofa chemically derivatized residue in place of a non-derivatized residue.

[0050] Chemical derivative refers to an Int6 protein or polypeptidehaving one or more residues chemically derivatized by reaction of afunctional side group. Examples of such derivatized molecules include,but are not limited to, those molecules in which free amino groups havebeen derivatized to form, for example, amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups. Free carboxyl groups may bederivatized to form salts, methyl and ethyl esters, or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzyllhistidine. Also included as chemicalderivatives are those proteins or peptides which contain one or morenaturally-occurring amino acid derivatives of the twenty standard aminoacids. For example, 4-hydroxyproline may be substituted for proline;5-hydroxylsine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

[0051] The nucleic acid sequences provided by the present invention areuseful as probes for a number of purposes. For example, they can be usedas probes in Southern hybridization to genomic DNA and in the RNaseprotection method for detecting point mutations. The probes can be usedto detect PCR amplification products. They may also be used to detectmismatches with the Int6 gene or mRNA using other techniques. Mismatchescan be detected using either enzymes (e.g., Si nuclease), chemicals(e.g., hydroxylamine or osmium tetroxide and piperidine), or changes inelectrophoretic mobility of mismatched hybrids as compared to totallymatched hybrids. These techniques are known in the art. The probes arecomplementary to Int6 gene coding sequences, although probes to intronsare also contemplated. An entire battery of nucleic acid probes is usedto compose a kit for detecting alteration of wild-type Int6 genes. Thekit allows for hybridization to the entire Int6 gene. The probes mayoverlap with each other or be contiguous.

[0052] If a riboprobe is used to detect mismatches with mRNA, it iscomplementary to the mRNA of the human wild-type Int6 gene. Theriboprobe thus is an anti-sense probe in that it does not code for theInt6 protein because it is of the opposite polarity to the sense strand.The riboprobe generally will be labeled with a radioactive,calorimetric, or fluorometric materials, which can be accomplished byany means known in the art. If the riboprobe is used to detectmismatches with DNA it can be of either polarity, sense or anti-sense.Similarly, DNA probes also may be used to detect mismatches.

[0053] Nucleic acid probes may also be complementary to mutant allelesof Int6 gene. These allele-specific probes are useful to detect similarmutations in other patients on the basis of hybridization rather thanmismatches. As mentioned above, the Int6 probes can also be used inSouthern hybridizations to genomic DNA to detect gross chromosomalchanges such as deletions and insertions. The probes can also be used toselect cDNA clones of Int6 genes from tumor and normal tissues. Inaddition, the probes can be used to detect Int6 mRNA in tissues todetermine if expression is altered as a result of mutation of wild-typeInt6 genes. Provided with the Int6 gene and the Int6 cDNA sequencesshown in SEQ ID NOs:1 and 3, design of particular probes is well withinthe skill of the ordinary artisan.

[0054] The present invention also relates to methods for detectingmutations of the Int6 gene in a subject.

[0055] For purposes of the present invention, subject means a mammal andmutation means inversion, translocation, insertion, deletion or pointmutation of the wild-type Int6 gene.

[0056] It is believed that many mutations found in tumor tissues will bethose leading to altered expression of Int6 protein. However, mutationsleading to non-functional gene products can also lead to cancer. It isfurther understood that point mutations can occur in regulatory regions(e.g. promoter) or can disrupt proper RNA processing thus leading toreduced Int6 mRNA expression or loss of expression of the Int6 proteinrespectively.

[0057] The fact that-integration of MMTV into the Int6 gene is observedin preneoplastic mouse mammary lesions suggests that mutations of Int6are involved in early events of cancer. The methods of detectingmutations of the Int6 gene can therefore provide diagnostic andprognostic information. For example, detection of mutation of the Int6gene in a tumor may effect the course of treatment chosen by aclinician. The methods of the present invention are applicable to anytumor in which mutations of Int6 occur. Loss of expression of the Int6gene has been observed in tumors of the lung and breast. Thus, these aretumors in which Int6 has a role in tumorigenesis. In addition, sinceInt6 is expressed in all tissues tested including brain, heart, kidney,liver, ovaries, spleen and testes, mutations affecting the expression ofthe Int6 gene may contribute to neoplasia in these tissues too. Finally,since lung and breast tumors are derived from epithelial tissue, themethods of the invention may be useful in the detection of epithelialcell derived cancers such as nonsmall cell lung carcinomas.

[0058] It is further understood by one skilled in the art that themethods for detection disclosed in the present invention can be usedprenatally to screen a fetus or presymptomatically to screen a subjectat risk of having cancer based on his/her family history.

[0059] In one embodiment of the invention, the method for detectingmutations of the Int6 gene comprises analyzing the DNA of a subject formutations of the wild-type Int6 gene. For analysis of DNA, a biologicalspecimen is obtained from the subject. Examples of biological specimensthat can be obtained for use in the present method, include, but are notlimited to, tissue biopsies and blood. Means for enriching a tissuepreparation for tumor cells are known in the art. For example, thetissue may be isolated from paraffin or cryostat sections. Cancer cellsmay also be separated from normal cells by flow cytometry. These as wellas other techniques for separating tumor from normal cells are wellknown in the art. Alternatively, primary cell cultures can beestablished from tumor biopsies using methods known to those of ordinaryskill in the art.

[0060] The DNA isolated from the biological specimen can be analyzed formutations of the Int6 gene by a variety of methods including Southernblotting after digestion with the appropriate restriction enzymes(restriction fragment length polymorphism, RFLP) (Botstein, D. Amer. J.Hum. Genet. (1980) 69:201-205, denaturing gradient electrophoresistechnique (Myers, R. M., Nature (1985) 313:495-498), oligonucleotidehybridization (Conner, R. et al., EMBO J. (1984) 3:13321-1326), RNasedigestion of a duplex between a probe RNA and the target DNA (Winter, E.et al., Proc. Natl. Acad. Sci. U.S.A. (1985) 82:7575-7579), polymerasechain reaction (PCR) (Saiki, P. K. et al., Science (1988) 239:487-491;U.S. Pat. Nos. 4,683,195 and 4,683,202), ligase chain reaction (LCR)(European Patent Application Nos. 0,320,308 and 0,439,182), andPCR-single stranded conformation analysis (PCR-SSCP) (Orita, M. et al.,Genomics (1989) 5:874-879; Dean, M. et al. Cell (1990) 61:863-871).

[0061] In one preferred embodiment, Southern blot analysis can be usedto examine tumor or blood DNA for gross rearrangement of the Int6 gene.The DNA to be analyzed via Southern analysis is digested with one ormore restriction enzymes. Following restriction digestion, resultant DNAfragments are separated by gel electrophoresis and the fragments aredetected by hybridization with a labelled nucleic acid probe (Southern,E.M. J. Mol. Biol. (1975) 98:503-517).

[0062] The nucleic acid sequence used as a probe in Southern analysiscan be labeled in single-stranded or double-stranded form. Labelling ofthe nucleic acid sequence can be carried out by techniques known to oneskilled in the art. Such labelling techniques can include radiolabelsand enzymes (Sambrook, J. et al. (1989) in “Molecular Cloning, ALaboratory Manual”, Cold Spring Harbor Press, Plainview, N.Y.). Inaddition, there are known non-radioactive techniques for signalamplification including methods for attaching chemical moieties topyrimidine and purine rings (Dale, R. N. K. et al. (1973) Proc. Natl.Acad. Sci., 70:2238-2242; Heck, R. F. 1968) S. Am. Chem. Soc.,90:5518-5523), methods which allow detection by chemiluminescence(Barton, S. K. et al. (1992) J. Am. Chem. Soc., 114:8736-8740) andmethods utilizing biotinylated nucleic acid probes (Johnson, T. K. etal. (1983) Anal. Biochem., 133:126-131; Erickson, P. F. et al. (1982) J.of Immunology Methods, 51:241-249; Matthaei, F. S. et al. (1986) Anal.Biochem., 157:123-128) and methods which allow detection by fluorescenceusing commercially available products. The size of the probe can rangefrom about 200 nucleotides to about several kilobases. A preferred probesize is about 500 to about 2000 nucleotides. The probe may be derivedfrom introns or exons of the Int6 gene. Each of the nucleic acidsequences used as a probe in Southern analysis is substantiallyhomologous to the corresponding portion of the murine or human Int6genes where these genes have the cDNA sequences shown in SEQ ID NOs:1 or3 respectively. In a preferred embodiment, the probes are derived from ahuman Int6 gene having the Int6 cDNA sequence shown in SEQ ID NO:3. By“substantially homologous” is meant a level of homology between thenucleic acid sequence used as a probe and the corresponding sequence ofthe human or murine Int6 genes. Preferably, the level of homology is inexcess of 70%, most preferably in excess of 80%, with a particularlypreferred nucleic acid sequence being in excess of 90% homologous withthe sequence of the mouse or human Int6 genes.

[0063] Once the separated DNA fragments are hybridized to the labellednucleic acid probes, the restriction digest pattern can be visualized byautoradiography and examined for the presence or absence of arestriction fragment length polymorphism (RFLP) associated with mutationof the Int6 gene.

[0064] In another preferred embodiment, genomic DNA may be analyzed formutations in the Int6 gene via PCR-SSCP. In this method, each of thepair of primers selected for use in PCR are designed to hybridize withsequences in the Int6 gene to permit amplification and subsequentdetection of mutations in the denatured amplification product vianon-denaturing polyacrylamide gel electrophoresis. In a preferredembodiment, primer pairs are derived from the human Int6 gene. In a morepreferred embodiment, primer pairs are derived from intronic sequenceswhich border the 5′ and 3′ ends of a given exon of the human Int6 gene.Examples of primer pairs permitting specific amplification of the exonsof the human Int6 gene include, but are not limited to,ACCAATAAAGTTTTAGTGAGCACAG SEQ ID NO:5 GCGCCCAAAGACCCCCTCAC SEQ ID NO:6TTAATCAGTTTCTTTGGGGA SEQ ID NO:7 AGTTTCTAATGACAAAACTTAC SEQ ID NO:8TCTTCTGCATTTTTAATTAG SEQ ID NO:9 CAAAATTAAGACGAGTTTAC SEQ ID NO:10CTTATTTTGTTTCTGTGGCC SEQ ID NO:11 CATGACAACTTTAAAATATTTTT SEQ ID NO:12AATTACAATGGGGTTTTAAA SEQ ID NO:13 GAAGAACCAAGGGAATCCTA SEQ ID NO:14TTCAAGAGTATTCACAATAT SEQ ID NO:15 TGTGAAAAAGACGAACTCAC SEQ ID NO:16AGTTTTCTTTATCTCACCCT SEQ ID NO:17 CAATATATATTTTAGTTTTAC SEQ ID NO:18CCGTTGACTTATTTTTACAG SEQ ID NO:19 AAATAAAAATTCACACTTAC SEQ ID NO:20TTGTTGTATTTGTACATATAG SEQ ID NO:21 ATCAAATCACGGTGTTCTTAC SEQ ID NO:22AAAACTAAGTTTTTAGGCCC SEQ ID NO:23 ATAGCTAACATAATACTCAC SEQ ID NO:24TTCCCTGTGTTTCCTTTTAG SEQ ID NO:25 ATAGAAGATGTGTGGTCTTAC SEQ ID NO:26GATTTCTTTTTGCATATTTTAG SEQ ID NO:27 CAAGAAAACTGACAGCAAGA SEQ ID NO:28

[0065] where SEQ ID NOs: 5 and 6 bound exon 1, SEQ ID NOs: 7 and 8 boundexon 2, SEQ ID NOs: 9 and 10 bound exon 3, SEQ ID NOs: 11 and 12 boundexon 4, SEQ ID NOs: 13 and 14 bound exon 5, SEQ ID NOs: 15 and 16 boundexon 6, SEQ ID NOs: 17 and 18 bound exon 8, SEQ ID NOs: 19 and 20 boundexon 9, SEQ ID NOs: 21 and 22 bound exon 10, SEQ ID NOs: 23 and 24 boundexon 11, SEQ ID NOs: 25 and 26 bound exon 12, and SEQ ID NOs: 27 and 28bound exon 13. Optimization of the amplification reaction to obtainsufficiently specific hybridization to Int6 gene sequences is wellwithin the skill in the art and is preferably achieved by adjusting theannealing temperature.

[0066] The primers of this invention can be synthesized using any of theknown methods of oligonucleotide synthesis (e.g., the phosphodiestermethod of Agarwal et al. 1972. Agnew. Chem. Int. Ed. Engl. 11:451, thephosphotriester method of Hsiung et al. 1979. Nucleic Acids Res. 6:1371,or the automated diethylphosphoramidite method of Beuacage et al. 1981.Tetrahedron Letters 22:1859-1862), or they can be isolated fragments ofnaturally occurring or cloned DNA. In addition, those skilled in the artwould be aware that oligonucleotides can be synthesized by automatedinstruments sold by a variety of manufacturers or can be commerciallycustom ordered and prepared. In one embodiment, the primers can bederivatized to include a detectable label suitable for detecting and/oridentifying the primer extension products (e.g., biotin, avidin, orradiolabeled dNTP's), or with a substance which aids in the isolation ofthe products of amplification (e.g. biotin or avidin).

[0067] In an alternative embodiment, primer pairs can be selected tohybridize to mutant forms of the Int6 disease gene. The selected primerpairs will hybridize sufficiently specifically to the mutated genesequences such that non-specific hybridization to wild-type Int6 genesequences will not prevent identification of the amplification productof the mutant gene sequence. Primer pairs which hybridize to mutationsin the Int6 gene sequence can be used to amplify specific mutant genesequences present in the DNA of a biological sample.

[0068] The amplification products of PCR can be detected either directlyor indirectly. Direct detection of the amplification products is carriedout via labelling of primer pairs. Labels suitable for labelling theprimers of the present invention are known to one skilled in the art andinclude radioactive labels, biotin, avidin, enzymes and fluorescentmolecules. The desired labels can be incorporated into the primers priorto performing the amplification reaction. A preferred labellingprocedure utilizes radiolabeled ATP and T4 polynucleotide kinase(Sambrook, J. et al. (1989) in “Molecular Cloning, A Laboratory Manual”,Cold Spring Harbor Press, Plainview, N.Y.). Alternatively, the desiredlabel can be incorporated into the primer extension products during theamplification reaction in the form of one or more labelled dNTPs. In thepresent invention, the labelled amplified PCR products can be analyzedfor mutations of the Int6 gene via separating the PCR products bynon-denaturing polyacrylamide gel electrophoresis, denaturingpolyacrylamide gel electrophoresis (PCR-SSCP) or via direct sequencingof the PCR-products.

[0069] In yet another embodiment, unlabelled amplification products canbe analyzed for mutations in the Int6 disease gene via hybridizationwith nucleic acid probes radioactively labelled or, labelled withbiotin, in Southern blots or dot blots. Nucleic acid probes useful inthe embodiment are those described earlier for Southern analysis. In yetanother embodiment, detection of point mutations may be accomplished bymolecular cloning of the allele present in the tumor tissue using thecDNA sequences set forth in SEQ ID NOs:1 and 3 and sequencing thatallele using techniques well known in the art.

[0070] In a second embodiment, the method for detecting mutations of theInt6 gene comprises analyzing the RNA of a subject for alterations inInt6-specific mRNA expression.

[0071] For the analysis of RNA by this method, RNA can be isolated fromblood or a tumor biopsy sample obtained from said subject where saidtumors include, but are not limited to, tumors of the breast and lung.

[0072] The RNA to be analyzed can be isolated from blood or tumor biopsysamples as whole cell RNA or as poly(A)⁺ RNA. Whole cell RNA can beisolated by methods known to those skilled in the art. Such methodsinclude extraction of RNA by differential precipitation (Birnbiom, H. C.(1988) Nucleic Acids Res., 16:1487-1497), extraction of RNA by organicsolvents (Chomczynski, P. et al. (1987) Anal. Biochem., 162:156-159) andextraction of RNA with strong denaturants (Chirgwin, J. M. et al. (1979)Biochemistry, 18:5294-5299). Poly(A)⁺ RNA can be selected from wholecell RNA by affinity chromatography on oligo-d(T) columns (Aviv, H. etal. (1972) Proc. Natl. Acad. Sci., 69:1408-1412). A preferred method ofisolating RNA is extraction of whole cell RNA by acid-phenol(Chomczynski et al. 1987).

[0073] The methods for analyzing the RNA for alterations in the patternor level of Int6-specific mRNA expression include Northern blotting(Alwine, J. C. et al. (1977) Proc. Natl. Acad. Sci., 74:5350-5354), dotand slot hybridization (Kafatos, F. C. et al. (1979) Nucleic Acids Res.,7:1541-1522), filter hybridization (Hollander, M. C. et al. (1990)Biotechniques; 9:174-179), RNase protection (Sambrook, J. et al. (1989)in “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Press,Plainview, N.Y.), reverse-transcription polymerase chain reaction(RTPCR) (Watson, J. D. et al. (1992) in “Recombinant DNA” SecondEdition, W.H. Freeman and Company, New York) and RT-PCR-SSCP. Onepreferred method is Northern blotting.

[0074] The nucleic acid sequence used as a probe for detectingInt6-specific mRNA expression is substantially homologous to SEQ. ID.NOs. 1 or 3. By “substantially homologous” is meant a level of homologybetween the nucleic acid sequence and the cDNA sequence of SEQ ID NOs:1or 3. Preferably, the level of homology is in excess of 70% morepreferably in excess on 80%, with a particularly preferred nucleic acidsequence being in excess of 90% homologous with the cDNA sequence shownin SEQ ID Nos. 1 or 3.

[0075] In a second preferred embodiment, the RNA is analyzed formutations in the Int6 gene by RT-PCR-SSCP. Single stranded cDNA isprepared from either tumor total RNA or polyA⁺ enriched RNA usingreverse transcriptase. In this method, each of the pairs of primersselected for use in PCR of the resultant single-stranded cDNA aredesigned to hybridize with sequences in the Int6 cDNA which are anappropriate distance apart (at least about 100-300 nucleotides) in thegene to permit amplification and subsequent detection of mutations inthe denatured amplification product via non-denaturing polyacrylamidegel electrophoresis. Primer pairs which can specifically hybridize tooverlapping Int6 gene sequences can be derived from the Int6 genesequence. Primer pairs can be derived from the cDNA sequences set forthin SEQ ID NOs 1 and 3. In a preferred embodiment, the primers arederived from the human Int6 cDNA sequence shown in SEQ ID NO:3. Eachprimer of a pair is a single-stranded oligonucleotide of about 15 toabout 20 bases in length which is complementary to a sequence at the 3′end of one of the strands of a double-stranded target sequence. Eachpair comprises two such primers, one of which is complementary 3′ endand the other of which is complementary to the other 3′ end of thetarget sequence. The target sequence is generally about 100 to about 300base pairs long. Optimization of the amplification reaction to obtainsufficiently specific hybridization to the Int6 cDNA is well within theskill in the art and is preferably achieved by adjusting the annealingtemperature. Alternatively, the denatured RT-PCR products can beanalyzed for mutations of the Int6 gene via direct sequencing of theRT-PCR products.

[0076] Yet another preferred method of analysis is the RNase protectionmethod, which is described in detail in Winter et al., Proc. Natl. Acad.Sci. USA, 82: p. 7575, (1985) and Meyers et al., Science, 230: p 1242,(1985). In the practice of the present invention the method involves theuse of a labeled riboprobe which is complementary to the human wild-typegene coding sequence. The riboprobe and either mRNA or DNA isolated fromthe tumor tissue are annealed (hybridized) together and subsequentlydigested with the enzyme RNase A which is able to detect mismatches in aduplex RNA structure. If a mismatch is detected by RNase A, it cleavesat the site of the mismatch. The riboprobe need not be the full lengthof the RNA or gene but can be a segment of either. If the riboprobecomprises only a segment of the Int6 RNA or Int6 gene it will bedesirable to use a number of these probes to screen the whole mRNAsequence for mismatches.

[0077] The present invention also encompasses recombinant proteinsderived from the cDNAs shown in SEQ ID Nos. 1 and 3. Recombinant Int6proteins can be produced by recombinant DNA methodology known to oneskilled in the art. Since the amino acid sequence of mouse (SEQ ID NO:2)and human (SEQ ID NO:4) Int6 proteins are identical, a suitable nucleicacid sequence capable of encoding a protein comprising all or part ofthe amino acid sequence shown in SEQ ID NO:4 is the sequence shown inSEQ ID NO:3 or in SEQ ID NO:1. In a preferred embodiment, such asuitable nucleic acid sequence can be cloned into a vector capable ofbeing transferred into, and replicated in, a host organism.

[0078] The vectors contemplated for use in the present invention includeany vectors into which a nucleic acid sequence as described above can beinserted, along with any preferred or required operational elements, andwhich vector can then be subsequently transferred into a host organismand replicated in such organism. Preferred vectors are those whoserestriction sites have been well documented and which contain theoperational elements preferred or required for transcription of thenucleic acid sequence.

[0079] The “operational elements” as discussed herein include at leastone promoter, at least one operator, at least one leader sequence, atleast one terminator codon, and any other DNA sequences necessary orpreferred for appropriate transcription and subsequent translation ofthe vector nucleic acid. In particular, it is contemplated that suchvectors will contain at least one origin of replication recognized bythe host organism along with at least one selectable marker and at leastone promoter sequence capable of initiating transcription of the nucleicacid sequence.

[0080] In construction of the cloning vector of the present invention,it should additionally be noted that multiple copies of the nucleic acidsequence and its attendant operational elements may be inserted intoeach vector. In such an embodiment, the host organism would producegreater amounts per vector of the desired Int6 protein. The number ofmultiple copies of the DNA sequence which may be inserted into thevector is limited only by the ability of the resultant vector due to itssize, to be transferred into and replicated and transcribed in anappropriate host microorganism.

[0081] In another embodiment, restriction digest fragments containing acoding sequence for Int6 protein can be inserted into a suitableexpression vector that functions in prokaryotic or eukaryotic cells. Bysuitable is meant that the vector is capable of carrying and expressinga complete nucleic acid sequence coding for Int6 protein. Examples ofexpression vectors that function in prokaryotic cells such as bacteriainclude, but are not limited to, T7 promoter based vectors and vectorsfor producing trpE and lacZ fusions. Preferred expression vectors arethose that function in a eukaryotic cell. Examples of such vectorsinclude but are not limited to, vaccinia virus vectors, adenovirus,herpesviruses, baculovirus or mammalian type C retroviral vectors.

[0082] The selected recombinant expression vector may then betransfected into a suitable eukaryotic cell system for purposes ofexpressing the recombinant protein. Such eukaryotic cell systemsinclude, but are not limited to cell lines such as human MCF10A or mouseHCll mammary epithelial cells. One preferred eukaryotic cell system foruse with baculovirus vectors is SF21 ovarian cells from spodopterafrugyerda.

[0083] The expressed recombinant protein may be detected by methodsknown in the art which include Coomassie blue staining and Westernblotting using sera containing anti-Int6 antibody.

[0084] In a further embodiment, the expressed recombinant protein can beobtained as a crude lysate or it can be purified by standard proteinpurification procedures known in the art which may include differentialprecipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis, affinity, andimmunoaffinity chromatography and the like. In the case ofimmunoaffinity chromatography, the recombinant protein may be purifiedby passage through a column containing a resin which has bound theretoantibodies specific for the Int6 protein.

[0085] In yet another embodiment, the present invention relates topeptides derived from the Int6 amino acid sequences shown in SEQ ID Nos.2 and 4 where those skilled in the art would be aware that the peptidesof the present invention, or analogs thereof, can be synthesized byautomated instruments sold by a variety of manufacturers, can becommercially custom ordered and prepared, or can be expressed fromsuitable expression vectors as described above. The term analog has beenpreviously described in the specification and for purposes of describingpeptides of the present invention, analogs can further include branchedor non-linear peptides. Preferred peptides are those that range fromabout 20 to about 24 amino acids length and are immunogenic. Asdescribed in Example 8 herein, mutation of the Int6 gene can result intruncation of the expressed Int6 protein. Based on previous studiesindicating that point-mutated ras proteins are processed in anappropriate manner to be targets for specific T cell-mediatedcytotoxicity (Tsang; K. Y. et al. (1994) Vaccine Research, 3:183-193;Takahashi, H. S.; et al. (1989) Science, 246:118-121, Jung, S.; et al.(1991) J. Exp. Med., 173:273-276, 1991; Peace, D. J.; et al. (1991) J.Immunol., 146:2059-2065; Fenton, R. G.; et al. (1993) J. Natl. CancerInst., 85:1294-1302; Gedde-Dahl, T., III; et al. (1992) Hum. Immunol.,33:266-274, and Gedde-Dahl, T., III; et al. (1992) Int. Immunol.,4:1331-1337), the mutated Int6 proteins may elicit a specific T-cellresponse via display on the tumor cell surface through the MHCapparatus.

[0086] The present invention therefore provides pharmaceuticalcompositions comprising Int6 protein or peptides derived therefrom foruse in vaccines and in immunotherapy methods. When used as vaccines toprotect mammals against cancer, the pharmaceutical composition cancomprise as an immunogen cell lysate from cells transfected with arecombinant expression vector or a culture supernatant containing theexpressed protein. Alternatively, the immunogen is a partially orsubstantially purified recombinant protein or a synthetic peptide.

[0087] The above compositions or formulations, both for veterinary andfor human use, comprise an immunogen as described above, together withone or more pharmaceutically acceptable carriers and optionally othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethod well-known in the pharmaceutical art.

[0088] All methods include the step of bringing into association theactive ingredient with the carrier which constitutes one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product into the desired formulation.

[0089] Formulations suitable for intravenous intramuscular,subcutaneous, or intraperitoneal administration conveniently comprisesterile aqueous solutions of the active ingredient with solutions whichare preferably isotonic with the blood of the recipient. Suchformulations may be conveniently prepared by dissolving solid activeingredient in water containing physiologically compatible substancessuch as sodium chloride (e.g. 0.1-2.0M), glycine, and the like, andhaving a buffered pH compatible with physiological conditions to producean aqueous solution, and rendering said solution sterile. These may bepresent in unit or multi-dose containers, for example, sealed ampoulesor vials.

[0090] The formulations of the present invention may incorporate astabilizer. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids which maybe used either on their own or as admixtures. These stabilizers arepreferably incorporated in an amount of 0.11-10,000 parts by weight perpart by weight of immunogen. If two or more stabilizers are to be used,their total amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. Informulating the immunogen of the present invention, anti-adsorptionagent may be used.

[0091] Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate theproteins, protein analogs or their functional derivatives, intoparticles of a polymeric material such as polyesters, polyamino acids,hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

[0092] When oral preparations are desired, the compositions may becombined with typical carriers, such as lactose, sucrose, starch, talcmagnesium stearate, crystalline cellulose, methyl cellulose,carboxymethyl cellulose, glycerin, sodium alginate or gum arabic amongothers.

[0093] The proteins of the present invention may be supplied in the formof a kit, alone, or in the form of a pharmaceutical composition asdescribed above.

[0094] Vaccination can be conducted by conventional methods. Forexample, the immunogen can be used in a suitable diluent such as salineor water, or complete or incomplete adjuvants. Further, the immunogenmay or may not be bound to a carrier to make the protein immunogenic.Examples of such carrier molecules include but are not limited to bovineserum albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid,and the like. The immunogen can be administered by any route appropriatefor antibody production such as intravenous, intraperitoneal,intramuscular, subcutaneous, and the like. The immunogen may beadministered once or at periodic intervals until a significant titer ofanti-Int6 antibody is produced. The antibody may be detected in theserum using an immunoassay as described below.

[0095] In yet another embodiment, the present invention providespharmaceutical compositions comprising nucleic acid sequence capable ofdirecting host organism synthesis of an Int6 protein or of a peptidederived from the Int6 protein sequence. Such nucleic acid sequence maybe inserted into a suitable expression vector by methods known to thoseskilled in the art. Expression vectors suitable for producing highefficiency gene transfer in vivo include, but are not limited to,retroviral, adenoviral and vaccinia viral vectors. Operational elementsof such expression vectors are disclosed previously in the presentspecification and are known to one skilled in the art. Such expressionvectors can be administered intravenously, intramuscularly,subcutaneously, intraperitoneally or orally.

[0096] Whether the immunogen is an Int6 protein, a peptide derivedtherefrom or a nucleic acid sequence capable of directing host organismsynthesis of Int6 protein or peptides derived therefrom, the immunogenmay be administered for either a prophylactic or therapeutic purpose.When provided prophylactically, the immunogen is provided in advance ofthe cancer or any symptom due to the cancer. The prophylacticadministration of the immunogen serves to prevent or attenuate anysubsequent onset of cancer. When provided therapeutically, the immunogenis provided at, or shortly after, the onset of cancer or any symptomassociated with the cancer.

[0097] When the immunogen is a partially or substantially purifiedrecombinant Into protein or an Int6 peptide, dosages effective to elicitan antibody response against Int6 range from about 10 μg to about 1500μg.

[0098] Dosages of Int6 protein—encoding nucleic acid sequence effectiveto elicit an antibody response against Int6 range from about 10 to about1000 μg.

[0099] The expression vectors containing a nucleic acid sequence capableof directing host organism synthesis of an Int6 protein(s) may besupplied in the form of a kit, alone, or in the form of a pharmaceuticalcomposition as described above.

[0100] The present invention further relates to a vaccine for immunizinga mammal against cancer comprising Int6 protein or an expression vectorcapable of directing host organism synthesis of Int6 protein in apharmaceutically acceptable carrier.

[0101] In addition to use as vaccines and in immunotherapy, the abovecompositions can be used to prepare antibodies to Int6 protein. Toprepare antibodies, a host animal is immunized using the Int6 protein orpeptides derived therefrom or aforementioned expression vectors capableof expressing Int6 protein or peptides derived therefrom. The host serumor plasma is collected following an appropriate time interval to providea composition comprising antibodies reactive with the virus particle.The gamma globulin fraction or the IgG antibodies can be obtained, forexample, by use of saturated ammonium sulfate or DEAE Sephadex, or othertechniques known to those skilled in the art. The antibodies aresubstantially free of many of the adverse side effects which may beassociated with other anti-viral agents such as drugs.

[0102] The antibody compositions can be made even more compatible withthe host system by minimizing potential adverse immune system responses.This is accomplished by removing all or a portion of the Fc portion of aforeign species antibody or using an antibody of the same species as thehost animal, for example, the use of antibodies from human/humanhybridomas. Humanized antibodies (i.e., nonimmunogenic in a human) maybe produced, for example, by replacing an immunogenic portion of anantibody with a corresponding, but nonimmunogenic portion (i.e.,chimeric antibodies). Such chimeric antibodies may contain the reactiveor antigen binding portion of an antibody from one species and the Fcportion of an antibody (nonimmunogenic) from a different species.Examples of chimeric antibodies, include but are not limited to,non-human mammal-human chimeras, rodent-human chimeras, murine-human andrat-human chimeras (Robinson et al., International Patent Application184,187; Taniguchi M., European Patent Application 171,496; Morrison etal., European Patent Application 173,494; Neuberger et al., PCTApplication WO 86/01533; Cabilly et al., 1987 Proc. Natl. Acad. Sci. USA84:3439; Nishimura et al., 1987 Canc. Res. 47:999; Wood et al., 1985Nature 314:446; Shaw et al., 1988 J. Natl. Cancer Inst. 80: 15553, allincorporated herein by reference).

[0103] General reviews of “humanized” chimeric antibodies are providedby Morrison S., 1985 Science 229:1202 and by Oi et al., 1986BioTechniques 4:214.

[0104] Suitable “humanized” antibodies can be alternatively produced byCDR or CEA substitution (Jones et al., 1986 Nature 321:552; Verhoeyan etal., 1988 Science 239:1534; Biedleret al. 1988 J. Immunol. 141:4053, allincorporated herein by reference).

[0105] The antibodies or antigen binding fragments may also be producedby genetic engineering. The technology for expression of both heavy andlight chain genes in E. coli is the subject the PCT patent applications;publication number WO 901443, WO901443, and WO 9014424 and in Huse etal., 1989 Science 246:1275-1281.

[0106] Alternatively, anti-Int6 antibodies can be induced byadministering anti-idiotype antibodies as immunogen. Conveniently, apurified anti-Int6 antibody preparation prepared as described above isused to induce anti-idiotype antibody in a host animal. The compositionis administered to the host animal in a suitable diluent. Followingadministration, usually repeated administration, the host producesanti-idiotype antibody. To eliminate an immunogenic response to the Fcregion, antibodies produced by the same species as the host animal canbe used or the FC region of the administered antibodies can be removed.Following induction of anti-idiotype antibody in the host animal, serumor plasma is removed to provide an antibody composition. The compositioncan be purified as described above for anti-Int6 antibodies, or byaffinity chromatography using anti-Int6 antibodies bound to the affinitymatrix. The anti-idiotype antibodies produced are similar inconformation to the authentic Int6 antigen and may be used to preparevaccine rather than using an Int protein.

[0107] When used as a means of inducing anti-Int6 antibodies in ananimal, the manner of injecting the antibody is the same as forvaccination purposes, namely intramuscularly, intraperitoneally,subcutaneously or the like in an effective concentration in aphysiologically suitable diluent with or without adjuvant. One or morebooster injections may be desirable.

[0108] For both in vivo use of antibodies to Int6 proteins andanti-idiotype antibodies and for diagnostic use, it may be preferable touse monoclonal antibodies. Monoclonal anti-Int6 antibodies oranti-idiotype antibodies can be produced by methods known to thoseskilled in the art. (Goding, J. W. 1983. Monoclonal Antibodies:Principles and Practice, Pladermic Press, Inc., NY, N.Y., pp. 56-97). Toproduce a human-human hybridoma, a human lymphocyte donor is selected. Adonor known to have the Int6 antigen may serve as a suitable lymphocytedonor. Lymphocytes can be isolated from a peripheral blood sample orspleen cells may be used if the donor is subject to splenectomy.Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes ora human fusion partner can be used to produce human-human hybridomas.Primary in vitro immunization with peptides can also be used in thegeneration of human monoclonal antibodies.

[0109] Antibodies secreted by the immortalized cells are screened todetermine the clones that secrete antibodies of the desired specificity.For monoclonal antibodies, the antibodies must bind to Int6 protein orpeptide. For monoclonal anti-idiotype antibodies, the antibodies mustbind to anti-Int6 antibodies. Cells producing antibodies of the desiredspecificity are selected.

[0110] The above described antibodies and antigen binding fragmentsthereof may be supplied in kit form alone, or as a pharmaceuticalcomposition for in vivo use. When used as a pharmaceutical compositionin immunotherapy, the antibodies or chimeric antibodies described hereinmay also be coupled to toxin molecules, radioisotopes, and drugs byconventional methods (Vitetta et al. (1991) in “Biologic Therapy ofCancer” De Vita V T, Hellman S., Rosenberg, S. A. (eds) J.B. LippincottCo. Philadelphia; Larson, S. M. et al. (1991) in “Biological Therapy ofCancer” De Vita V.T., Hellman S., Rosenberg, S. A. (eds) J.B. LippincottCo., Philadelphia). Examples of toxins to which the antibodies may becoupled to include, but are not limited to, Ricin and pseudomonasendotoxin. Examples of drugs or chemotherapeutic agents include, but arenot limited to, adriamycin. Examples of radioisotopes, include, but arenot limited to, ¹³¹I. Antibodies covalently conjugated to theaforementioned agents can be used in cancer immunotherapy. Theantibodies may also be used as an immunoaffinity agent to purify Int6proteins, for therapeutic uses, and for diagnostic use in immunoassaysas described below.

[0111] The present invention therefore relates to a third method fordetecting mutations of the Int6 gene comprising:

[0112] analyzing the protein of a subject for alterations in Int6protein expression.

[0113] For analysis of protein by this method, protein is obtained frombiological specimens such as tumor biopsy samples and the like. Theprotein can be obtained as a crude lysate or it can be further purifiedby methods known to one skilled in the art (Sambrook, J. et al. (1989)in “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor press,Plainview, N.Y.).

[0114] Crude protein lysate can be analyzed for Int6 protein byimmunoassays using anti-Int6 antibody.

[0115] Immunoassays of the present invention may be a radioimmunoassay,Western blot assay, immunofluorescent assay, enzyme immunoassay,chemiluminescent assay, immunohistochemical assay and the like. Standardtechniques known in the art for ELISA are described in Method inImmunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley andSons, 1980 and Campbell et al., Methods of Immunology, W.A. Benjamin,Inc., 1964, both of which are incorporated herein by reference. Suchassays may be a direct, indirect, competitive, or noncompetitiveimmunoassay as described in the art. (Oellerich, M. 1984. J. Clin. Chem.Clin. BioChem. 22:895-904).

[0116] Detection of the Int6 protein anti-Int6 antibody complex formedcan be accomplished by reaction of the complex with a secondary antibodysuch as labelled anti-rabbit antibody. The label may be an enzyme whichis detected by incubating the complex in the presence of a suitablefluorimetric or calorimetric reagent. Other detectable labels may alsobe used, such as radiolabels, or colloidal gold, and the like. Thelabelled Int6 protein-anti-Int6 antibody complex is then visualized byautoradiography.

[0117] The present invention also includes a method for treating cancercomprising administering pharmaceutical compositions comprising an Int6protein, an expression vector containing nucleic acid sequence capableof directing host organism synthesis of an Int6 protein, or anti-Int6antibody in a therapeutically effective amount. When providedtherapeutically, the Int6 protein, Int6 protein-encoding expressionvector or anti-Int6 antibody coupled to radioisotopes, toxin moleculesor drugs is provided at (or shortly after) the onset of infection or atthe onset of any symptom of infection or disease caused by cancer. Thetherapeutic administration of the Int6 protein, Int6 protein-encodingexpression vector, or anti-Int6 antibody serves to attenuate theinfection or disease.

[0118] The present invention also includes another method for treating asubject having cancer comprising:

[0119] (a) immunizing a subject with an amount of Int6 protein or anexpression vector capable of directing host organism synthesis of Int6protein effective to elicit a specific T cell response;

[0120] (b) isolating said T cells from said immunized subject; and

[0121] (c) administering said T cells to said immunized subject or to anunimmunized subject in a therapeutically effective amount.

[0122] T cells populations reactive against the Int6 protein may beisolated from a peripheral blood sample or spleen cells of a donorimmunized with the Int6 protein. Epstein-Barr virus (EBV) can be used toimmortalize human lymphocytes or a human fusion partner can be used toproduce human-human hybridomas. Primary in vitro immunization with Int6protein can also be used in the generation of T cells reactive to theInt6 protein.

[0123] T cells are cultured for about 7 to about 90 days (Yanelli, J. R.J. Immunol. Methods 139:1-16 (1991)) and then screened to determine theclones of the desired reactivity against the other peptide contained inthe immunogenic chimeric protein using known methods of assaying T cellreactivity; T cells producing the desired reactivity are thus selected.

[0124] The above described T cells may be used for in vivo use astreatment for individuals afflicted with cancer by administering T cellsto a mammal intravenously, intraperitoneally, intramuscularly orsubcutaneously. Preferred routes of administration are intravenously orintraperitoneally.

[0125] The present invention also relates to a gene therapy method inwhich an expression vector containing a nucleic acid sequencerepresenting the wild-type Int6 gene is administered to a subject havinga mutation of the Int6 gene. A nucleic acid sequence representingwild-type Int6 gene is that shown in SEQ ID No. 1 and SEQ ID No:3. Suchnucleic acid sequence may be inserted into a suitable expression vectorby methods known to those skilled in the art. Expression vectorssuitable for producing high efficiency gene transfer in vivo includeretroviral, adenoviral and vaccinia viral vectors.

[0126] Expression vectors containing a nucleic acid sequencerepresenting wild-type Int6 gene can be administered intravenously,intramuscularly, subcutaneously, intraperitoneally or orally. Apreferred route of administration is intravenously.

[0127] The invention also provides a diagnostic kit for detectingmutations of the Int6 gene. This diagnostic kit comprises purified andisolated nucleic acid sequences useful as PCR primers in analyzing DNAor RNA for mutations of the Int6 disease gene.

[0128] Any articles or patents referenced herein are incorporated byreference. The following examples are presented to illustrate variousaspects of the invention but are in no way intended to limit the scopethereof.

EXAMPLES Example 1 Identification of a Novel Integration Site for theMMTV Genome

[0129] To identify new Int loci in mammary tumor DNAs that would not bebiased by selective inbreeding for a high cancer phenotype, a feralmouse strain derived from a single breeding pair of M. musculus trappedin Czechoslovakia (CZECH II) (Gallahan, D. and Callahan, R. (1987)J.Virol, 61:66-74) was utilized. The CZECH II mice, unlikehigh-incidence inbred mouse strains, lack endogenous MMTV genomes but docontain an infectious strain of MMTV that is transmitted congenitallythrough the milk (Callahan, R. et al. (1982) Proc. Natl. Acad. Sci.U.S.A., 79:4113-4117). In the CZECH II mice, several preneoplastichyperplastic outgrowth lines (HOGs) were developed which are stable,clonal-dominant and develop focal mammary tumors which sometimesmetastasis to the lung. In order to detect the integration of MMTVproviral genome into genomic DNA of the HOGs or HOG-derived tumor orlung metastasis from a mouse having a HOG-derived tumor, cellular DNAwas isolated from a CZECH II HOG line designated CZZ1 or from CZZ-1derived malignant tumors or lung metastasis and analyzed by Southernblotting as follows. 10 ug of cellular DNA was digested by EcoRI, run ona 0.8% agarose gel, transferred to a nylon membrane and hybridized withMMTV LTR probe as described in Gallahan, D. and Callahan R. (J. Virol(1987) 61:66-74. In Brief, before hybridization, filters were soaked for4 to 24 hours at 37° C. in a prehybridization solution containing 3×SSPE (1× SSPE is 180 mM NaCl, 10 mM NaH₂PO₄ [pH 7.4], and 1 mM EDTA), 5×Denhardt solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02%bovine serum albumin), 2.5% dextran sulfate, and 50% formamide.Denatured probe was added to a solution similar to the prehybridizationsolution except that it contained 40% formamide. The mixture was addedto the plastic bag containing prehybridized filters and incubated at 37°C. for 24 h. The filters were washed in stringent conditions involvingthree changes (each change done after 20 to 30 minutes of washing) of0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.5%sodium dodecyl sulfate at 65° C. The filters were exposed to Kodak XAR-5C-ray film overnight or for up to several days. The results of theseSouthern blots are shown in FIGS. 1A and 1B. The results presented inFIG. 1A show that DNA from one CZZ-1 HOG line contained three MMTVproviral genomes (six EcoRI restriction fragments) integrated into thecellular DNA and that these proviral genomes were also present inprimary tumors which arose independently from within the HOG. Theresults further demonstrated that CZZ-1 was clonal-dominant populationof preneoplastic cells because these six MMTV-related fragments werereproducibly found in the original outgrowth and in each succeedingtransplant generation. Indeed, the clonal-dominant nature of the CZZ-1HOG is illustrated by tumor 1262 (lane 3, FIG. 1A) in which one completeproviral genome, corresponding to the 4.8 and 2.8 Kb EcoRI restrictionfragments, has been lost. The observation that five out of the thirteentumor DNAs analyzed in FIG. 1A and five out of eleven independentmetastatic lesions in the lung of the mouse bearing tumor 4973 (FIG. 1B)contained additional integrated MMTV genomes suggested that theadditional integration events may have contributed to tumor progressionby activating (or inactivating) additional cellular genes. However,since none of the known common insertions sites (Wnt-1, Fgf-3, Int-3,Wnt-3, and Fgf-4) were rearranged by MMTV in the CZZ-1 HOG, recombinantclones were obtained for each of the EcoRI host-MMTV junctionrestriction fragments in the CZZ-1 HOG and subclones of the hostsequences were used as probes to screen Southern blots of independentMMTV-induced mammary tumor DNAs for evidence of MMTV-induced DNArearrangements.

[0130] Using this approach, a probe consisting of the host sequencesflanking one of the MMTV proviruses in the CZZ-1 HOG (i.e., the 3.0 Kbfragment shown in FIG. 1A) was used to identify an Int6 commonintegration site in the CZZ-1 HOG derived tumor 22 (FIG. 2A, lanes 2 and3) and independent mammary tumors 1139 (FIG. 2B, lanes 2 and 3) and 3144(FIG. 2C, lanes 2 and 3). In brief, cellular DNAs (10 μg) from eachtumor DNA and from normal liver DNA (lane 1 in each panel) were digestedwith EcoRI, run on a 0.8% agarose gel, and then blotted onto a nylonmembrane. Lanes 1 and 2 of the blots in FIGS. 2A and 2B were hybridizedwith probe D which corresponds to host flanking sequences. Lane 2 ofFIGS. 2A and 2B was subsequently hybridized with MMTV gag sequences(Gallahan and Callahan (1987) J. Virol, 61:66-74) and the results areshown in lane 3 of FIGS. 2A and 2B. Lanes 1 and 2 in FIG. 2C werehybridized with probe C. Lane 2 was subsequently hybridized with MMTVenv sequences (Gallahan and Callahan (1987) J. Virol, 61:66-74) and theresult is shown in lane 3 of FIG. 2C. Hybridizations of the respectiveblots with the first probe were carried out as described earlier in thisExample (Gallahan and Callahan ((1987) J. Virol. 61:66-74). Where blotswere subsequently hybridized with a second probe, the blots werereprobed according to Gallahan and Callahan ((1987) J. Virol., 61:66-74)as follows. In brief, Genetran filters were stripped of probe DNA byplacing the filters in 100 ml of 0.4 N NaOH and incubated for 20 minutesat room temperature. This solution was then discarded and replaced with0.1 M Tris (pH 7.5)-0.1× SSC-0.5% sodium dodecyl sulfate solution toneutralize the filter. The filter was incubated for 15 minutes at roomtemperature, after which the neutralization solution was replaced andincubation was continued for another 15 minutes. The filter was thenprehybridized and hybridized as described earlier in this Example.Genetran filters treated in this way could be re-used at least fivetimes before noticeable DNA loss.

[0131] The results show that MMTV-induced rearrangements (indicated byan arrow in FIGS. 2A-2C) were detected in the two independentMMTV-induced mammary tumor DNAs (lanes 2 and 3 of FIGS. 2B and 2C). Ofinterest, in each case where MMTV-induced rearrangements were detected,the rearranged restriction fragment co-migrated with a fragmentcontaining either MMTV gag or env sequences. These results demonstratethat the transcriptional orientation of the viral genome in each tumorwas in the same direction. Thus, the host sequences adjacent to theintegrated MMTV genome define a new common integration site from MMTVdesignated Int6.

Example 2 Isolation of the Murine Int6 Gene

[0132] To obtain recombinant genomic clones of the Int6 loci, a subcloneof the 3.0 kb EcoRI fragment (shown in FIG. 1A) containing the hostflanking sequences (i.e. probe D, FIG. 3) was used to probe a lambdaphage library (Stratagene, Lajolla, Calif.) of genomic DNA from mousestrain 129/sv. This genomic DNA contains wild-type Int6 DNA asdetermined by nucleotide sequence analysis. An additional threeoverlapping lambda clones which span 47 kb of the Int6 locus have alsobeen obtained using probes A-C (FIG. 3). Together, these fouroverlapping lambda clones span the murine Int6 gene as shown in FIG. 3.The gene is located centromeric of the myc proto-oncogene on mousechromosome 15. Further nucleotide sequence analysis of the genomicclones of the Int6 locus demonstrated that the gene contains thirteenexons which span 34 Kb of genomic DNA as shown in FIG. 3.

Example 3 Expression of the Int6 Gene In CZECH II Mammary Tumors Toexamine Int6 expression in CZECH II mammary tumors, total RNA (20 μg)prepared from the Int6 negative tumor (tumor 178) and from Int6 positivetumors having viral insertions in Int6 (22 and 1139) was denatured inthe presence of formaldehyde and run on a 1% agarose gel containingformaldehyde. The RNA was then transferred to a nylon membrane andhybridized with probe A (FIG. 3) as described in Gallahan, D. andCallahan, R. (1987) J. Virol., 61:66-74. The results of the Northernblots are shown in FIG. 4. In brief, hybridization with probe A detecteda 1.4 Kb species of RNA in each of three tumors and tumor 1139 alsocontained a 0.9 Kb species of RNA related to sequences in probe A. Theobservation that tumor 178 (FIG. 4) and several other MMTV inducedmammary tumors in which Int6 is not rearranged by the virus expressedthe 1.4 kb RNA species detected by probe A. This suggests that in tumors22 and 1139 the level of the Int6 gene expression is not an importantconsequence of viral integration in this locus. Example 4 Isolation ofWild-Type Murine Int6 cDNA

[0133] In order to isolate a cDNA corresponding to wild-type 1.4 kb Int6RNA, a murine cDNA library of an MMTV induced mammary tumor in whichInt6 was not rearranged by MMTV was prepared using standard techniques(Sambrook et al (1989) in “Molecular Cloning, A Laboratory Manual”, ColdSpring Harbor Press, Plainview N.Y.) and was probed with probe A (probeA is the XbaI fragment shown in FIG. 3). The nucleotide sequence of themurine Int6 cDNA was determined and is shown in FIG. 5. Translation ofthe 1.4 kb species of Int6 RNA revealed an open reading frame whichencodes a 43.5 kilodalton protein. As shown in FIG. 5, this proteincontains two potential N-glycosylation site motifs (Gavel, Y. and vonHeijne, G. (1990) Protein Eng., 3:433-442) as well as potentialphosphorylation site motifs for cyclic AMP/cyclic GMP-dependent proteinkinase (Glass, D. B. et al (1986) J. Biol. Chem., 261:2987-2993),protein kinase C (Kishimoto, A. et al (1985) J. Biol. Chem.,260:12492-12499), tyrosine kinases (Hunter, T. and Cooper, J. A. (1985)Ann. Rev. Biochem, 54:897-930) and casein kinase II (Pinna, L. A. (1990)Biochim Biophys Acta, 1054:267-284).

Example 5 Conservation of the Int6 cDNA Across Species

[0134] Since all of the genes whose expression has been affected oraltered by MMTV integration in mouse mammary tumors have been highlyconserved through evolution (Dickson, C. and Peters, G. (1987) Nature326:883; Rijsewizk, K. F. et al (1987) Cell, 50:649-657; Robbins, J. etal (1992) J. Virol., 66:2594-2599), the conservation of the Int6 gene ingenomic DNA of different species was examined via Southern blotanalysis. Cellular DNAs (10 μg each) from C. elegans, Drosophila,Xenopus, chicken, mouse, and human were digested with BamH1, run on a0.8% agarose gel, transferred to a nylon membrane and hybridizedovernite with Int6 cDNA in 3x SSPE (1× SSPE is 180 mM BaCl, 10mMNaH₂PO₄[pH 7.4, and 1 mM EDTA), 5× Denhardt solution (0.02% Ficoll,0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 5% dextransulfate and 2% SDS (sodium dodecyl sulfate) at 65° C. Afterhybridization, the blot was washed in stringent condition involvingthree changes (each change done after 20 to 30 minutes of washing) 0.5×SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) plus 0.5% SDS at65° C. Exposure to Kodak XAR-5 film was for three days. As shown in FIG.6, Int6 related sequences can be detected in all the eukaryotic speciesexamined. Restriction fragments of yeast DNA which contain Int6 relatedsequences were also detected by Southern blot analysis (data not shown).

[0135] In addition, the nucleotide sequence of a cDNA clone of theDrosophila homolog of Int6 has been determined (data not shown) and thededuced amino acid sequence of the Drosophila Int6 protein is 60%identical to the human/mouse deduced amino acid sequences. Takentogether, these results demonstrate an extensive evolutionaryconservation of the Int6 gene which is indicative of Int6 serving abasic life function.

Example 6 Detection of Int6-Specific mRNA Expression in Target Tissues

[0136] Having detected the 1.4 Kb Int6 RNA species in a tumor in whichthe gene was not rearranged by MMTV (i.e., tumor 178, FIG. 4) normaladult tissues and embryos at different stages of development weresurveyed for Int6 RNA expression via Northern analysis as follows. TotalRNAs (10 μg each) was prepared from the indicated tissues. The RNAs weredenatured in the presence of formaldehyde and run on 1% agarose gelscontaining formaldehyde. The RNA samples were then transferred to anylon membrane and hybridized sequentially with a β-actin probe and thenan Int6 cDNA probe under conditions described in Gallahan and Callahan(J. Virol., 61:66-74 (1987)). As shown in FIG. 7, Int6 RNA is expressedin all adult tissues tested, including the mammary gland, and expressionof Int6 RNA in embryos was detected as early as day eight ofdevelopment.

Example 7 Detection of Int6-Specific mRNA Expression in Target Tissues

[0137] To detect Int6-specific mRNA expression, Random primed cDNA isprepared using total RNA from the various tissues. A 700 bp fragment ofInt6 murine cDNA is amplified by PCR using the primers shown as SEQ IDNO.29 TGTCCACATATTCTACGCTA and SEQ ID NO. 30 TGTATGTCATCCTTTATACA. Theconditions of the PCR are: 30 cycles of denaturation at 94° C. for 1minute, annealing at 55° C. for 1 minute, and extension at 72° C. for 2minutes. After the last cycle there is an additional extension time of 2minutes. The PCR product is run on a 1.0% agarose gel. The RT-PCRproducts are detected by photographing the gel following staining withethidium bromide.

Example 8 Rearrangement of Int6 by MMTV Leads to the Expression of NovelSpecies of Int6 RNA in Mammary Tumors

[0138] Inspection of the mapping data for the murine Int6 gene as shownin FIG. 3 reveals that all of the viral integration events occur withinintrons of the Int6 gene and that the transcriptional direction of theintegrated viral genome is in the opposite orientation to that of theInt6 gene. There are at least four possible scenarios for the role MMTVplays at the Int6 locus. In the first, the Int6 gene is in fact not thetarget gene on which MMTV acts. At the Wntl, Fgf-3, and Fgf-4 loci(Dickson, C., et al. (1984) Cell. 37:529-536; Nusse, R., et al. (1982)Cell. 31:99-109; Peters, G., et al. (1989) Proc. Natl. Acad. Sci. USA.86:5678-5682), MMTV integration sites cluster around the target gene inan ordered fashion and the transcriptional orientation of the integratedviral genomes 5′ of the target gene are in the opposite direction tothat of the target gene whereas those 3′ of the target gene are in thesame transcriptional orientation. Further, based on published reports,MMTV integration sites are within 15 kb of the particular target gene.However, since no evidence was found for the activation of expression ofRNA corresponding to sequences up to 13 kb 3′ of the Int6 gene, thelocation of the putative target gene would have to be more than 24 kbfrom the integrated MMTV genome in CZZ-1 HOG and more than 30 kb intumor 1139 (see FIG. 3), thereby making this possibility much lesslikely.

[0139] A second possible consequence of MMTV integration into the Int6gene is the activation of expression of a new chimeric RNA transcript ofInt6 initiated by the 3′ LTR of the integrated viral genome. Ananalogous situation occurs in MMTV-induced rearrangements of Int-3(Robbins, J., et al. (1992) J. Virol., 66:2594-2599) except in the caseof Int6, this would result in the expression of Int6 anti-sense RNAsince the transcriptional orientation of the viral genome is opposite tothat of Int6. Such a result would represent a trans-dominant mutationthat would inactivate the expression of both alleles. However, using avariety of techniques, MMTV-induced Int6 antisense RNA expression hasnot been detected.

[0140] A third possibility assumes that the Int6 gene is the target forMMTV integration within the Int6 locus. In this case the viral insertionwould disrupt the expression of one allele and reveal the presence of aspontaneous recessive mutation in the other allele. To examine thispossibility the nucleotide sequence of cDNA corresponding to thenon-rearranged allele of Int6 in tumor 22 and tumor 1139 was determinedand in both cases no mutation was found.

[0141] The fourth and more plausible scenario is that MMTV integrationinto the Int6 gene causes the expression of a biologically activatedgene product (like Int3, (Robbins, J., et al. (1992) J. Virol.,66:2594-2599)) or a dominant-negative gene product either of whichderegulates the normal control of mammary epithelial cellular growthleading to hyperplasia of the affected mammary epithelial cells. Thiswould create a premalignant epithelial cell population with whichmammary tumors could subsequently develop.

[0142] To test whether integration of MMTV into the Int6 gene producesaltered RNA species, the nucleotide sequence of cDNA clones of Int6 RNAfrom tumor 1139 and tumor 22. In each case transcription of therearranged allele resulted in the expression of a chimeric RNA specieswhich terminated at a cryptic transcription stop signal in the reverseU3 portion of the MMTV LTR (FIG. 8). Of interest, a similar cryptictranscription termination signal has previously been shown to be activein certain MMTV induced rearrangements Fgf-3(Clausse, N., (1993)Virology, 194:157-165).

[0143]FIGS. 9A and 9B show the nucleotide sequences of Int6-MMTV LTR RNAspecies detected in tumors 1139 (FIG. 9A) and 22 (FIG. 9B). In FIG. 9A,two RNA species from the rearranged allele (FIG. A, 900 bp and 965 bp)were detected. The RNA species correspond to the 900 bp RNA speciesdetected by Northern blot analysis in FIG. 4. In one RNA species exon 5was spliced to the end of the U5 portion of the MMTV LTR and in theother species splicing occurred at a cryptic splice acceptor site inintron 5. Similarly, FIG. 9B, CZZ-1 there were three chimeric RNAspecies in which exon 9 was spliced to one of three different crypticsplice acceptor sites in intron 9. Since the size of the chimeric tumorRNA species is similar to that of the normal Int6 RNA, they wentundetected in the Northern blot analysis of tumor 22 RNA FIG. 4.

[0144] Translation of the rearranged Int6 RNA species into putativeproteins revealed that from all five species, the product is a truncatedchimera of the Int6 amino acid sequences linked to novel amino acidsequences encoded by an Int6 intron and/or reverse MMTV LTR nucleotidesequences. The presence of the 965 bp RNA species in tumor 1139 suggeststhat truncation of the Int6 gene product is an important consequence ofMMTV integration.

Example 9 Isolation Of The Human Int6 Gene

[0145] Mouse Int6 cDNA was used to probe a cDNA library of human lungRNA in lambda phage (Clontech Inc.) and using primers specific for thehuman Int6 cDNA (SEQ ID NO:3) cloned from the human lung cDNA library, aPI phage library of human genomic DNA was screened by PCR for anInt6-related clone by Genome Systems Inc.(St. Louis, Mo.). Using thisapproach, a Pi phage clone and two lambda clones containing over 50 kbof human genomic DNA were obtained. Nucleotide sequence analysis ofthese clones revealed that the human Int6 gene is organized as shown inFIG. 10. As in the mouse genome, the human Int6 gene is composed of 13exons. The human Int6 gene also contains a CA-repeat sequence in theseventh intron (FIG. 10).

Example 10 Size Polymorphism of the CA Repeat Sequence Contained inIntron 7

[0146] Primers complementary to the nucleic acid sequences flanking theCA-repeat in intron 7 of the human Int6 gene (FIG. 1) and having thesequences shown in SEQ ID NO:31 GTGAAAATGACATGAAATTTCAG and SEQ ID NO:32TGCAGTGTGACAATATGGGC were used to PCR amplify the portion of the Int6gene containing the CA-repeat in human genomic DNA. The conditions ofthe PCR were: 30 cycles of denaturation at 94° C. for one minute,annealing at 55° C. for one minute, and extension at 72° C. for twominutes. After the last cycle, there was an additional extension time oftwo minutes. Separation of the PCR products via non-denaturing 6%polyacrylamide gel electrophoresis revealed that 46 of the 84 individualDNAs analyzed were heterozygous (informative) for different sizealleles.

Example 11 Chromosomal Localization of the Human Int 6 Gene

[0147] To determine the chromosomal localization of the human Int6 gene,Southern blot analysis of human DNA and DNA isolated from rodent-humansomatic cell hybrids containing individual human chromosomes wasperformed. In brief, rodent-human somatic cell hybrid DNA or human DNAwas digested with HindIII and hybridized with human Int6 cDNA at 37° C.for 24 hours in 3× SSPE, 5× Denhardt solution, 2.5% dextran sulfate and40% formamide. The blot was then washed under stringent conditionsinvolving three changes (each change done after twenty to thirtyminutes) of 0.1× SSC and 0.5% SDS at 65° C. The results of this Southernblot are shown in FIG. 12 where lanes 1 and 2 contain Chinesehamster-human somatic cell hybrid DNA containing human chromosome 6(lane 1) or 8 (lane 2), lane 3 contains DNA from mouse-human somaticcell hybrids containing human chromosomes 3, 7, 8, 15 and 17 and lane 4contains human genomic DNA. The 23 kb fragment shown in lane 4 containsan Int6-related pseudogene and the coding sequences of the Int6 gene aredefined by the 4.5, 3.5 and 3.0 kb fragments shown in lane 4. Theresults show that while the 23 kb fragment was detected only in lane 1(hybrids containing human chromosome 6), the 4.5 and 3.5 kb fragmentswere detected in lane 2 (hybrids containing human chromosome 8). Inaddition, the 3.0 kb human fragment (see lane 4) was also detected inboth lanes 1 and 2. However, the detection of the 3.0 kb fragment inlane 3 confirmed that the 3.0 kb fragment contains human Int6-relatedsequence rather than Chinese hamster sequence since mouse DNA was shownnot to contain an Int6-related 3.0 kb Hind III fragment. These resultsdemonstrate that the human Int6 gene is located on chromosome 8. Thischromosomal localization was confirmed and further refined to chromosome8q22-q24 by linkage analysis using the CA-repeat polymorphism describedin Example 10.

Example 12 Detection of Mutations in the Int6 Gene in Human Breast TumorDNA Resulting in a Loss of Heterozygosity

[0148] Based on the results in MMTV-induced mouse mammary tumorspresented earlier, DNA from human breast cancer biopsies was analyzed todetermine whether the Int6 gene might be a target for mutation duringmalignant progression in human breast cancer. In brief, DNA from primaryhuman breast tumor and from matching normal tissue of 40 individualsthat were informative at the Int6 CA-repeat sequence were analyzed forevidence of loss of heterozygosity (LOH) in the tumor DNA via the PCRmethod utilized in Example 10. In eleven of these tumors (25%), therewas either a complete loss or a significantly reduced signal of oneallele relative to the matching normal DNA. Five of these eleven tumorDNAs (T) and their matching normal samples (L) are shown in FIG. 13.These results show that Int6 is a target for mutation during malignantprogression in human breast cancer. Further, since Int6 gene expressionhas been detected in all tissues analyzed (see FIG. 7), mutation of theInt6 gene may occur during progression of cancers found in othertissues.

Example 13 Analysis of the Human Int6 Gene for Mututions by PCR-SSCPUsing Primer Pairs Derived From Sequences Bounding Exons of the Int6Gene

[0149] Primer pairs complementary to nucleic acid sequences bounding 12of the 13 human Int6 exons (FIG. 14) are shown as SEQ ID NOs:5-28. Usingprimer pairs selected from SEQ ID NOs:5-28, each of the exons of theInt6 gene except exon 7 were amplified from the eleven primary breasttumor DNAs having loss of heterozygosity at the Int6 gene (see Example12) and from DNA of matching normal tissue samples by PCR and the PCRproducts were analyzed for single-stranded conformation polymorphism(SSCP) via denaturing polyacrylamide gel electrophoresis. Exon 7 wasanalyzed by hybrid mismatch methodology using the portion of human Int6cDNA corresponding to exon 7. Analysis by both single strandconformation polymorphism analysis (SSCP) and hybrid mismatchmethodologies of the primary breast tumor DNAs having loss ofheterozygosity at the Int6 gene detected no point mutations in theremaining allele (data not shown). However, the promoter and intronregions of the remaining allele were not analyzed for mutations. Ofcourse, if mutations of the promoter, intron and/or coding regions ofthe Int6 gene were detected in tumor samples, these mutations could beconfirmed by nucleotide sequence analysis of the variant allele in thetumor and matching normal tissue DNA.

Example 14 Loss of Expression of Int6 mRNA in Human Breast and LungTumor Samples

[0150] Total RNA isolated from 36 malignant human breast tumors, 2preneoplastic lesions (high grade dysplasia) and matching normal tissuewere surveyed for Int6 mRNA expression via Northern blot analysis usingsequential hybridization with β-actin and human Int6 cDNA probes underconditions described in Gallahan and Callahan (J. Virol. 61:66-74(1987)). Of these 38 samples, 14 (37%) exhibited significantly reducedor nondetectable Int6 mRNA expression as compared with matching normaltissue samples.

[0151] Of these 14 cases, 8 were invasive ductal carcinomas (IDC), 2were comedocarcinomas (IDC with a prevalent intraductal component), 2were lobular carcinomas and 2 were the preneoplastic lesions. Arepresentative Northern blot is shown in FIG. 15.

[0152] In a separate study, Int6 mRNA expression in 47 nonsmall celllung carcinomas (NSCLC) and matching normal tissues was surveyed viaNorthern analysis of 10 μg total RNA as described above in this Example.A representative Northern blot is shown in FIG. 16 and the results ofthe Northern analyses of the 47 NSCLC samples are summarized in Table 1.TABLE 1 ASSOCIATIONS BETWEEN LOSS OF EXPRESSION OF Int6 AND CLINICALPARAMETERS RE- DUCED OR NON- DETECT- ABLE NORMAL TOTAL EXPRES- EXPRES-SAM- P HISTOTYPE SION SION PLES VALUE SQUAMOUS CELL 3 (14%) 18 21ADENOCARCINOMA 8 (67%) 4 12 0.003 BRONCHIOLOALVEOLAR 2 (33%) 4 6

[0153] Of interest, 14 of 47 NSCLCs showed loss of Int6 mRNA expressionand loss of Int6 mRNA expression was observed to have a highlysignificant association (P=0.003) with a particular histologic subtype(Table 1, adenocarcinoma of NSCLC).

[0154] The results presented in this Example are consistent with theconclusion that loss of Int6 expression is an early event in humanbreast cancer and a contributing factor in other neoplasias such asthose of the lung.

Example 15 Characterization of the Murine Int6 Protein

[0155] The Int6 gene encodes a 50 kD protein with three potentialtranslation start sites (at 27 bp, 174 bp and 189 bp of the mousesequence). Each of these polypeptides were detected as products of invitro translation of Int6 RNA in the rabbit reticulocyte system. Rabbitpolyclonal sera to synthetic peptides corresponding to amino acidresidues 57-71 (peptide 47) and 262-281 (peptide 20) of mouse Int6 wereprepared and used to immunoprecipitate the Int6 in vitro translationproducts. This immunoprecipitation was competed by the correspondingpeptide. Western blot analysis of protein extracts of adult mousetissues (brain, lung, kidney, muscle, heart and mammary gland), usingantibodies to peptide 20 (rAB 20), detected a major 40 KD polypeptide aswell as a triplet of 80 KD polypeptides. Reaction with thesepolypeptides was competed with peptide 20. Of interest, expression of a30 KD Int6 related polypeptide was also observed in Western blots ofsalivary gland extracts. It is believed that the 40 KD polypeptide mayrepresent a cleavage product of the 50 KD precursor and that the 80 KDcross reacting polypeptide may represent dimers of the 40 KDpolypeptides.

[0156] In addition, cell fractionation studies and immunofluorescencestudies using antibodies to the above peptides have demonstrated thatInt6 is primarily localized to the cytoplasm. Moreover, inimmunofluorescence studies of mouse embryos, the Int6 protein waslocalized to the golgi apparatus.

[0157] Finally, Int6 protein, which contains three candidate proteinkinase C (PKC) phosphorylation sites, was expressed in bacteria usingthe QUIA Express Type 4 vector (Quiagen, Chatsworth Calif.), purifiedand shown to be phosphorylated by PKC (data not shown).

1 32 1 1505 DNA Murine INT6 1 aacaagcgct cctttccccc ggcaagatggcggagtacga cctgactact cgcatcgcgc 60 attttctgga tcggcacctg gtctttccgcttcttgagtt tctctctgtg aaagagattt 120 ataatgaaaa agaattatta caaggaaaattagatcttct tagtgatacc aatatggtgg 180 actttgctat ggatgtttac aaaaacctttattctgatga tatccctcat gctttgagag 240 aaaaaagaac cacagttgtt gcgcagctgaaacagctcca ggcagaaaca gaaccaattg 300 tgaagatgtt tgaagatcca gaaactacaaggcagatgca gtcaaccagg gatggcagga 360 tgttatttga ctacctggca gacaaacatgggtttaggca agagtactta gatacactct 420 acagatacgc aaaattccag tatgagtgtggaaattactc tggagctgca gagtatcttt 480 acttctttag agttttggtc ccagcaacagatagaaatgc tttaagttcg ctctggggaa 540 aactggcctc tgaaatctta atgcagaattgggatgcagc catggaagac cttactcgat 600 taaaagaaac catagacaat aattctgtgagttctccact ccagtctctt cagcagcgaa 660 catggctcat tcattggtct ctatttgtttttttcaacca tccaaagggc cgtgataaca 720 ttattgatct cttcctttac caaccacagtatcttaatgc aattcagaca atgtgtccac 780 atattctacg ctatttgact actgccgtcataaccaacaa agatgtgcgg aaacgccggc 840 aggtgctgaa agatctggtg aaagtgattcaacaggagtc ttacacatat aaagacccaa 900 ttacagaatt tgttgaatgc ctatatgttaactttgattt tgacggggct cagaaaaagc 960 tgagagaatg tgaatcagtg ctcgtgaatgacttcttcct ggtagcgtgt ctggaggact 1020 tcattgagaa tgcccgtctc ttcatatttgagacgttttg tcgtatccac cagtgtatca 1080 gcattaatat gttagcagat aaactgaatatgactccaga agaagctgaa agatggattg 1140 tgaatttgat tagaaatgcg aggttggatgccaagattga ttctaaacta ggtcatgtgg 1200 taatgggcaa caatgcagtc tcgccctaccagcaagtgat tgaaaagacc aaaagccttt 1260 cttttagaag ccaaatgttg gccatgaatattgaaaagaa acttaatcag aacagtagat 1320 cagaggctcc caactgggca acccaagactctggcttcta ttaaaggatt ataaagaaaa 1380 gaagaaaaag gaataagtga aagacacagtagccattgtg tataaaggat gacatacatt 1440 tttagaagca attaacatgt ttgctacaaattttggagaa tttgaataaa attggctatg 1500 attaa 1505 2 396 PRT Murine INT6 2Met Val Asp Phe Ala Met Asp Val Tyr Lys Asn Leu Tyr Ser Asp Asp 1 5 1015 Ile Pro His Ala Leu Arg Glu Lys Arg Thr Thr Val Val Ala Gln Leu 20 2530 Lys Gln Leu Gln Ala Glu Thr Glu Pro Ile Val Lys Met Phe Glu Asp 35 4045 Pro Glu Thr Thr Arg Gln Met Gln Ser Thr Arg Asp Gly Arg Met Leu 50 5560 Phe Asp Tyr Leu Ala Asp Lys His Gly Phe Arg Gln Glu Tyr Leu Asp 65 7075 80 Thr Leu Tyr Arg Tyr Ala Lys Phe Gln Tyr Glu Cys Gly Asn Tyr Ser 8590 95 Gly Ala Ala Glu Tyr Leu Tyr Phe Phe Arg Val Leu Val Pro Ala Thr100 105 110 Asp Arg Asn Ala Leu Ser Ser Leu Trp Gly Lys Leu Ala Ser GluIle 115 120 125 Leu Met Gln Asn Trp Asp Ala Ala Met Glu Asp Leu Thr ArgLeu Lys 130 135 140 Glu Thr Ile Asp Asn Asn Ser Val Ser Ser Pro Leu GlnSer Leu Gln 145 150 155 160 Gln Arg Thr Trp Leu Ile His Trp Ser Leu PheVal Phe Phe Asn His 165 170 175 Pro Lys Gly Arg Asp Asn Ile Ile Asp LeuPhe Leu Tyr Gln Pro Gln 180 185 190 Tyr Leu Asn Ala Ile Gln Thr Met CysPro His Ile Leu Arg Tyr Leu 195 200 205 Thr Thr Ala Val Ile Thr Asn LysAsp Val Arg Lys Arg Arg Gln Val 210 215 220 Leu Lys Asp Leu Val Lys ValIle Gln Gln Glu Ser Tyr Thr Tyr Lys 225 230 235 240 Asp Pro Ile Thr GluPhe Val Glu Cys Leu Tyr Val Asn Phe Asp Phe 245 250 255 Asp Gly Ala GlnLys Lys Leu Arg Glu Cys Glu Ser Val Leu Val Asn 260 265 270 Asp Phe PheLeu Val Ala Cys Leu Glu Asp Phe Ile Glu Asn Ala Arg 275 280 285 Leu PheIle Phe Glu Thr Phe Cys Arg Ile His Gln Cys Ile Ser Ile 290 295 300 AsnMet Leu Ala Asp Lys Leu Asn Met Thr Pro Glu Glu Ala Glu Arg 305 310 315320 Trp Ile Val Asn Leu Ile Arg Asn Ala Arg Leu Asp Ala Lys Ile Asp 325330 335 Ser Lys Leu Gly His Val Val Met Gly Asn Asn Ala Val Ser Pro Tyr340 345 350 Gln Gln Val Ile Glu Lys Thr Lys Ser Leu Ser Phe Arg Ser GlnMet 355 360 365 Leu Ala Met Asn Ile Glu Lys Lys Leu Asn Gln Asn Ser ArgSer Glu 370 375 380 Ala Pro Asn Trp Ala Thr Gln Asp Ser Gly Phe Tyr 385390 395 3 1500 DNA Homo sapiens 3 actccctttt ctttggcaag atggcggagtacgacttgac tactcgcatc gcgcactttt 60 tggatcggca tctagtcttt ccgcttcttgaatttctctc tgtaaaggag atatataatg 120 aaaaggaatt attacaaggt aaattggaccttcttagtga taccaacatg gtagactttg 180 ctatggatgt atacaaaaac ctttattctgatgatattcc tcatgctttg agagagaaaa 240 gaaccacagt ggttgcacaa ctgaaacagcttcaggcaga aacagaacca attgtgaaga 300 tgtttgaaga tccagaaact acaaggcaaatgcagtcaac cagggatggt aggatgctct 360 ttgactacct ggcggacaag catggttttaggcaggaata tttagataca ctctacagat 420 atgcaaaatt ccagtacgaa tgtgggaattactcaggagc agcagaatat ctttattttt 480 ttagagtgct ggttccagca acagatagaaatgctttaag ttcactctgg ggaaagctgg 540 cctctgaaat cttaatgcag aattgggatgcagccatgga agaccttaca cggttaaaag 600 agaccataga taataattct gtgagttctccacttcagtc tcttcagcag agaacatggc 660 tcattcactg gtctctgttt gttttcttcaatcaccccaa aggtcgcgat aatattattg 720 acctcttcct ttatcagcca caatatcttaatgcaattca gacaatgtgt ccacacattc 780 ttcgctattt gactacagca gtcataacaaacaaggatgt tcgaaaacgt cggcaggttc 840 taaaagatct agttaaagtt attcaacaggagtcttacac atataaagac ccaattacag 900 aatttgttga atgtttatat gttaactttgactttgatgg ggctcagaaa aagctgaggg 960 aatgtgaatc agtgcttgtg aatgacttcttcttggtggc ttgtcttgag gatttcattg 1020 aaaatgcccg tctcttcata tttgagactttctgtcgcat ccaccagtgt atcagcatta 1080 acatgttggc agataaattg aacatgactccagaagaagc tgaaaggtgg attgtaaatt 1140 tgattagaaa tgcaagactg gatgccaagattgattctaa attaggtcat gtggttatgg 1200 gtaacaatgc agtctcaccc tatcagcaagtgattgaaaa gaccaaaagc ctttccttta 1260 gaagccagat gttggccatg aatattgagaagaaacttaa tcagaatagc aggtcagagg 1320 ctcctaactg ggcaactcaa gattctggcttctactgaag aaccataaag aaaagatgaa 1380 aaaaaaaact atcaaagaaa gatgaaataataaaactatt atataaaggg tgacttacat 1440 tttggaaaca acatattacg tataaattttgaagaattgg aataaaattg attcatttta 1500 4 396 PRT Homo sapiens 4 Met ValAsp Phe Ala Met Asp Val Tyr Lys Asn Leu Tyr Ser Asp Asp 1 5 10 15 IlePro His Ala Leu Arg Glu Lys Arg Thr Thr Val Val Ala Gln Leu 20 25 30 LysGln Leu Gln Ala Glu Thr Glu Pro Ile Val Lys Met Phe Glu Asp 35 40 45 ProGlu Thr Thr Arg Gln Met Gln Ser Thr Arg Asp Gly Arg Met Leu 50 55 60 PheAsp Tyr Leu Ala Asp Lys His Gly Phe Arg Gln Glu Tyr Leu Asp 65 70 75 80Thr Leu Tyr Arg Tyr Ala Lys Phe Gln Tyr Glu Cys Gly Asn Tyr Ser 85 90 95Gly Ala Ala Glu Tyr Leu Tyr Phe Phe Arg Val Leu Val Pro Ala Thr 100 105110 Asp Arg Asn Ala Leu Ser Ser Leu Trp Gly Lys Leu Ala Ser Glu Ile 115120 125 Leu Met Gln Asn Trp Asp Ala Ala Met Glu Asp Leu Thr Arg Leu Lys130 135 140 Glu Thr Ile Asp Asn Asn Ser Val Ser Ser Pro Leu Gln Ser LeuGln 145 150 155 160 Gln Arg Thr Trp Leu Ile His Trp Ser Leu Phe Val PhePhe Asn His 165 170 175 Pro Lys Gly Arg Asp Asn Ile Ile Asp Leu Phe LeuTyr Gln Pro Gln 180 185 190 Tyr Leu Asn Ala Ile Gln Thr Met Cys Pro HisIle Leu Arg Tyr Leu 195 200 205 Thr Thr Ala Val Ile Thr Asn Lys Asp ValArg Lys Arg Arg Gln Val 210 215 220 Leu Lys Asp Leu Val Lys Val Ile GlnGln Glu Ser Tyr Thr Tyr Lys 225 230 235 240 Asp Pro Ile Thr Glu Phe ValGlu Cys Leu Tyr Val Asn Phe Asp Phe 245 250 255 Asp Gly Ala Gln Lys LysLeu Arg Glu Cys Glu Ser Val Leu Val Asn 260 265 270 Asp Phe Phe Leu ValAla Cys Leu Glu Asp Phe Ile Glu Asn Ala Arg 275 280 285 Leu Phe Ile PheGlu Thr Phe Cys Arg Ile His Gln Cys Ile Ser Ile 290 295 300 Asn Met LeuAla Asp Lys Leu Asn Met Thr Pro Glu Glu Ala Glu Arg 305 310 315 320 TrpIle Val Asn Leu Ile Arg Asn Ala Arg Leu Asp Ala Lys Ile Asp 325 330 335Ser Lys Leu Gly His Val Val Met Gly Asn Asn Ala Val Ser Pro Tyr 340 345350 Gln Gln Val Ile Glu Lys Thr Lys Ser Leu Ser Phe Arg Ser Gln Met 355360 365 Leu Ala Met Asn Ile Glu Lys Lys Leu Asn Gln Asn Ser Arg Ser Glu370 375 380 Ala Pro Asn Trp Ala Thr Gln Asp Ser Gly Phe Tyr 385 390 3955 25 DNA Artificial Sequence Oligonucleotide primer 5 accaataaagttttagtgag cacag 25 6 20 DNA Artificial Sequence Oligonucleotide primer6 gcgcccaaag accccctcac 20 7 20 DNA Artificial Sequence Oligonucleotideprimer 7 ttaatcagtt tctttgggga 20 8 22 DNA Artificial SequenceOligonucleotide primer 8 agtttctaat gacaaaactt ac 22 9 20 DNA ArtificialSequence Oligonucleotide primer 9 tcttctgcat ttttaattag 20 10 20 DNAArtificial Sequence Oligonucleotide primer 10 caaaattaag acgagtttac 2011 20 DNA Artificial Sequence Oligonucleotide primer 11 cttattttgtttctgtggcc 20 12 23 DNA Artificial Sequence Oligonucleotide primer 12catgacaact ttaaaatatt ttt 23 13 20 DNA Artificial SequenceOligonucleotide primer 13 aattacaatg gggttttaaa 20 14 20 DNA ArtificialSequence Oligonucleotide primer 14 gaagaaccaa gggaatccta 20 15 20 DNAArtificial Sequence Oligonucleotide primer 15 ttcaagagta ttcacaatat 2016 20 DNA Artificial Sequence Oligonucleotide primer 16 tgtgaaaaagacgaactcac 20 17 20 DNA Artificial Sequence Oligonucleotide primer 17agttttcttt atctcaccct 20 18 21 DNA Artificial Sequence Oligonucleotideprimer 18 caatatatat tttagtttta c 21 19 20 DNA Artificial SequenceOligonucleotide primer 19 ccgttgactt atttttacag 20 20 20 DNA ArtificialSequence Oligonucleotide primer 20 aaataaaaat tcacacttac 20 21 21 DNAArtificial Sequence Oligonucleotide primer 21 ttgttgtatt tgtacatata g 2122 21 DNA Artificial Sequence Oligonucleotide primer 22 atcaaatcacggtgttctta c 21 23 20 DNA Artificial Sequence Oligonucleotide primer 23aaaactaagt ttttaggccc 20 24 20 DNA Artificial Sequence Oligonucleotideprimer 24 atagctaaca taatactcac 20 25 20 DNA Artificial SequenceOligonucleotide primer 25 ttccctgtgt ttccttttag 20 26 21 DNA ArtificialSequence Oligonucleotide primer 26 atagaagatg tgtggtctta c 21 27 22 DNAArtificial Sequence Oligonucleotide primer 27 gatttctttt tgcatatttt ag22 28 20 DNA Artificial Sequence Oligonucleotide primer 28 caagaaaactgacagcaaga 20 29 20 DNA Artificial Sequence Oligonucleotide primer 29tgtccacata ttctacgcta 20 30 20 DNA Artificial Sequence Oligonucleotideprimer 30 tgtatgtcat cctttataca 20 31 23 DNA Artificial SequenceOligonucleotide primer 31 gtgaaaatga catgaaattt cag 23 32 20 DNAArtificial Sequence Oligonucleotide primer 32 tgcagtgtga caatatgggc 20

We claim:
 1. A purified and isolated Int6 gene.
 2. A purified andisolated Int6 gene comprising a nucleic acid sequence according to SEQID NO:
 3. 3. A purified and isolated Int6 gene encoding a protein havingan amino acid sequence according to SEQ ID No.
 4. 4. A cDNA having asequence according to SEQ ID NO.
 3. 5. The cDNA of claim 4, said cDNAhaving ATCC deposit numbers 97029 and
 97030. 6. A method of assaying asample comprising contacting said sample with at least one nucleotidesequence derived from the Int6 gene.
 7. The method of claim 6, whereinsaid step of assaying comprises using said nucleotide sequence as aprobe in Southern blot analysis.
 8. The method of claim 7, wherein saidprobe used is derived from wild-type Int6 gene sequence.
 9. The methodof claim 8, wherein said sequence is cDNA.
 10. The method of claim 9,wherein said cDNA comprises the sequence according to SEQ ID NO:3. 11.The method of claim 6, wherein said step of assaying comprises usingsaid nucleotide sequence as a probe in Northern blot analysis.
 12. Themethod of claim 11, wherein said probe is derived from a cDNA having asequence according to SEQ ID NO.
 3. 13. The method of claim 6, whereinsaid step of assaying comprises using said nucleotide sequences as PCRprimers.
 14. The method of claim 13, wherein said primers are derivedfrom wild-type Int6 gene sequence.
 15. The method of claim 14, whereinsaid step of assaying comprises using said primers in PCR-SSCP analysis.16. The method of claim 15, wherein said primers are selected from SEQID NOs: 5 thru SEQ ID NOs:
 28. 17. The method of claim 14, wherein saidsequence is a cDNA sequence according to SEQ ID NO:3.
 18. The method ofclaim 17, wherein said step of assaying comprises using said primers inRT-PCR analysis.
 19. The method of claim 17, wherein said step ofassaying comprises using said primers in RT-PCR-SSCP analysis. 20.Purified and isolated primers derived from Int6 gene sequence, saidprimers being capable of specifically hybridizing to Int6 gene sequence.21. The primers of claim 20, where said sequence is intronic sequence ofthe wild-type Tnt6 gene.
 22. The primers of claim 21, wherein saidprimers have the sequences shown in SEQ ID NOs: 5 to 28 and in SEQ IDNOs: 31 and
 32. 23. A diagnostic kit useful for assaying a sample, saidkit comprising: primers having nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 5 thru 28 and SEQ ID NOs: 31 and
 32. 24.The primers of claim 20, wherein said sequence is a cDNA.
 25. Theprimers of claim 24, wherein said cDNA has a sequence according to SEQID NO:3.
 26. A diagnostic kit useful for assaying a sample comprising:at least one nucleic acid sequence derived from an Int6 gene having acoding sequence shown in SEQ ID NO:3, said nucleic acid sequence beingcapable of specifically hybridizing to the Int6 gene.
 27. A method ofassaying a sample comprising contacting said sample with antibodydirected against Int6 protein or against peptide fragments derivedtherefrom.
 28. The method of claim 27, wherein said step of assayingcomprises immunohistochemical assay
 29. A recombinant Int6 proteinhaving an amino acid sequence according to SEQ ID NO:4, or a peptidefragment thereof.
 30. A purified and isolated nucleic acid sequencecapable of directing host organism synthesis of Int6 protein or apeptide derived therefrom.
 31. A recombinant expression vectorcomprising the nucleic acid sequence of claim
 30. 32. The recombinantexpression vector of claim 31, wherein said nucleic acid sequences iscontained in SEQ ID NO:3.
 33. A pharmaceutical composition comprisingthe recombinant expression vector of claim
 30. 34. Antibodies havingspecific binding affinity for Int6 protein or peptides derivedtherefrom.
 35. The antibodies of claim 34, wherein said antibodies aremonoclonal antibodies.
 36. A pharmaceutical composition comprising theantibodies of claim 34 coupled to a toxin, radionucleotide or drug. 37.A pharmaceutical composition comprising the recombinant Int6 protein ofclaim
 29. 38. A vaccine comprising the recombinant protein of claim 28in a pharmaceutically acceptable carrier.
 39. A vaccine comprising therecombinant expression vector of claim 31 in a pharmaceuticallyacceptable carrier.
 40. A method of immunotherapy for a subject havingcancer, said method comprising administering to said subject in aneffective amount a pharmaceutical composition according to claim
 33. 41.A method of immunotherapy for a subject having cancer, said methodcomprising administering to said subject in an effective amount apharmaceutical composition according to claim
 36. 42. A method ofimmunotherapy for a subject having cancer, said method comprisingadministering to said subject in an effective amount a pharmaceuticalcomposition according to claim
 37. 43. A host cell transformed ortransfected with the recombinant vector of claim
 31. 44. The host cellof claim 43, wherein said cell is prokaryotic.
 45. The host cell ofclaim 43, wherein said cell is eukaryotic.